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Sample records for homology modeled proteins

  1. Protein homology model refinement by large-scale energy optimization.

    Science.gov (United States)

    Park, Hahnbeom; Ovchinnikov, Sergey; Kim, David E; DiMaio, Frank; Baker, David

    2018-03-20

    Proteins fold to their lowest free-energy structures, and hence the most straightforward way to increase the accuracy of a partially incorrect protein structure model is to search for the lowest-energy nearby structure. This direct approach has met with little success for two reasons: first, energy function inaccuracies can lead to false energy minima, resulting in model degradation rather than improvement; and second, even with an accurate energy function, the search problem is formidable because the energy only drops considerably in the immediate vicinity of the global minimum, and there are a very large number of degrees of freedom. Here we describe a large-scale energy optimization-based refinement method that incorporates advances in both search and energy function accuracy that can substantially improve the accuracy of low-resolution homology models. The method refined low-resolution homology models into correct folds for 50 of 84 diverse protein families and generated improved models in recent blind structure prediction experiments. Analyses of the basis for these improvements reveal contributions from both the improvements in conformational sampling techniques and the energy function.

  2. Accurate protein structure modeling using sparse NMR data and homologous structure information.

    Science.gov (United States)

    Thompson, James M; Sgourakis, Nikolaos G; Liu, Gaohua; Rossi, Paolo; Tang, Yuefeng; Mills, Jeffrey L; Szyperski, Thomas; Montelione, Gaetano T; Baker, David

    2012-06-19

    While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining (1)H(N), (13)C, and (15)N backbone and (13)Cβ chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2-1.9 Å relative to the conventional determined NMR ensembles and of 0.9-1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.

  3. Illustrating and homology modeling the proteins of the Zika virus [version 2; referees: 2 approved

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    Sean Ekins

    2016-09-01

    Full Text Available The Zika virus (ZIKV is a flavivirus of the family Flaviviridae, which is similar to dengue virus, yellow fever and West Nile virus. Recent outbreaks in South America, Latin America, the Caribbean and in particular Brazil have led to concern for the spread of the disease and potential to cause Guillain-Barré syndrome and microcephaly. Although ZIKV has been known of for over 60 years there is very little in the way of knowledge of the virus with few publications and no crystal structures. No antivirals have been tested against it either in vitro or in vivo. ZIKV therefore epitomizes a neglected disease. Several suggested steps have been proposed which could be taken to initiate ZIKV antiviral drug discovery using both high throughput screens as well as structure-based design based on homology models for the key proteins. We now describe preliminary homology models created for NS5, FtsJ, NS4B, NS4A, HELICc, DEXDc, peptidase S7, NS2B, NS2A, NS1, E stem, glycoprotein M, propeptide, capsid and glycoprotein E using SWISS-MODEL. Eleven out of 15 models pass our model quality criteria for their further use. While a ZIKV glycoprotein E homology model was initially described in the immature conformation as a trimer, we now describe the mature dimer conformer which allowed the construction of an illustration of the complete virion. By comparing illustrations of ZIKV based on this new homology model and the dengue virus crystal structure we propose potential differences that could be exploited for antiviral and vaccine design. The prediction of sites for glycosylation on this protein may also be useful in this regard. While we await a cryo-EM structure of ZIKV and eventual crystal structures of the individual proteins, these homology models provide the community with a starting point for structure-based design of drugs and vaccines as well as a for computational virtual screening.

  4. Chemical shift homology in proteins

    International Nuclear Information System (INIS)

    Potts, Barbara C.M.; Chazin, Walter J.

    1998-01-01

    The degree of chemical shift similarity for homologous proteins has been determined from a chemical shift database of over 50 proteins representing a variety of families and folds, and spanning a wide range of sequence homologies. After sequence alignment, the similarity of the secondary chemical shifts of C α protons was examined as a function of amino acid sequence identity for 37 pairs of structurally homologous proteins. A correlation between sequence identity and secondary chemical shift rmsd was observed. Important insights are provided by examining the sequence identity of homologous proteins versus percentage of secondary chemical shifts that fall within 0.1 and 0.3 ppm thresholds. These results begin to establish practical guidelines for the extent of chemical shift similarity to expect among structurally homologous proteins

  5. A restraint molecular dynamics and simulated annealing approach for protein homology modeling utilizing mean angles

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    Maurer Till

    2005-04-01

    Full Text Available Abstract Background We have developed the program PERMOL for semi-automated homology modeling of proteins. It is based on restrained molecular dynamics using a simulated annealing protocol in torsion angle space. As main restraints defining the optimal local geometry of the structure weighted mean dihedral angles and their standard deviations are used which are calculated with an algorithm described earlier by Döker et al. (1999, BBRC, 257, 348–350. The overall long-range contacts are established via a small number of distance restraints between atoms involved in hydrogen bonds and backbone atoms of conserved residues. Employing the restraints generated by PERMOL three-dimensional structures are obtained using standard molecular dynamics programs such as DYANA or CNS. Results To test this modeling approach it has been used for predicting the structure of the histidine-containing phosphocarrier protein HPr from E. coli and the structure of the human peroxisome proliferator activated receptor γ (Ppar γ. The divergence between the modeled HPr and the previously determined X-ray structure was comparable to the divergence between the X-ray structure and the published NMR structure. The modeled structure of Ppar γ was also very close to the previously solved X-ray structure with an RMSD of 0.262 nm for the backbone atoms. Conclusion In summary, we present a new method for homology modeling capable of producing high-quality structure models. An advantage of the method is that it can be used in combination with incomplete NMR data to obtain reasonable structure models in accordance with the experimental data.

  6. Homology modeling and protein engineering of alkane monooxygenase in Burkholderia thailandensis MSMB121: in silico insights.

    Science.gov (United States)

    Jain, Chakresh Kumar; Gupta, Money; Prasad, Yamuna; Wadhwa, Gulshan; Sharma, Sanjeev Kumar

    2014-07-01

    The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism.

  7. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

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    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

  8. A discriminative method for family-based protein remote homology detection that combines inductive logic programming and propositional models.

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    Bernardes, Juliana S; Carbone, Alessandra; Zaverucha, Gerson

    2011-03-23

    Remote homology detection is a hard computational problem. Most approaches have trained computational models by using either full protein sequences or multiple sequence alignments (MSA), including all positions. However, when we deal with proteins in the "twilight zone" we can observe that only some segments of sequences (motifs) are conserved. We introduce a novel logical representation that allows us to represent physico-chemical properties of sequences, conserved amino acid positions and conserved physico-chemical positions in the MSA. From this, Inductive Logic Programming (ILP) finds the most frequent patterns (motifs) and uses them to train propositional models, such as decision trees and support vector machines (SVM). We use the SCOP database to perform our experiments by evaluating protein recognition within the same superfamily. Our results show that our methodology when using SVM performs significantly better than some of the state of the art methods, and comparable to other. However, our method provides a comprehensible set of logical rules that can help to understand what determines a protein function. The strategy of selecting only the most frequent patterns is effective for the remote homology detection. This is possible through a suitable first-order logical representation of homologous properties, and through a set of frequent patterns, found by an ILP system, that summarizes essential features of protein functions.

  9. Homology modeling, molecular docking and DNA binding studies of nucleotide excision repair UvrC protein from M. tuberculosis.

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    Parulekar, Rishikesh S; Barage, Sagar H; Jalkute, Chidambar B; Dhanavade, Maruti J; Fandilolu, Prayagraj M; Sonawane, Kailas D

    2013-08-01

    Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.

  10. GPCR-SSFE: A comprehensive database of G-protein-coupled receptor template predictions and homology models

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    Kreuchwig Annika

    2011-05-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs transduce a wide variety of extracellular signals to within the cell and therefore have a key role in regulating cell activity and physiological function. GPCR malfunction is responsible for a wide range of diseases including cancer, diabetes and hyperthyroidism and a large proportion of drugs on the market target these receptors. The three dimensional structure of GPCRs is important for elucidating the molecular mechanisms underlying these diseases and for performing structure-based drug design. Although structural data are restricted to only a handful of GPCRs, homology models can be used as a proxy for those receptors not having crystal structures. However, many researchers working on GPCRs are not experienced homology modellers and are therefore unable to benefit from the information that can be gleaned from such three-dimensional models. Here, we present a comprehensive database called the GPCR-SSFE, which provides initial homology models of the transmembrane helices for a large variety of family A GPCRs. Description Extending on our previous theoretical work, we have developed an automated pipeline for GPCR homology modelling and applied it to a large set of family A GPCR sequences. Our pipeline is a fragment-based approach that exploits available family A crystal structures. The GPCR-SSFE database stores the template predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs. Users are able to browse the GPCR dataset according to their pharmacological classification or search for results using a UniProt entry name. It is also possible for a user to submit a GPCR sequence that is not contained in the database for analysis and homology model building. The models can be viewed using a Jmol applet and are also available for download along with the alignments. Conclusions The data provided by GPCR-SSFE are useful for investigating

  11. GPCR-SSFE: a comprehensive database of G-protein-coupled receptor template predictions and homology models.

    Science.gov (United States)

    Worth, Catherine L; Kreuchwig, Annika; Kleinau, Gunnar; Krause, Gerd

    2011-05-23

    G protein-coupled receptors (GPCRs) transduce a wide variety of extracellular signals to within the cell and therefore have a key role in regulating cell activity and physiological function. GPCR malfunction is responsible for a wide range of diseases including cancer, diabetes and hyperthyroidism and a large proportion of drugs on the market target these receptors. The three dimensional structure of GPCRs is important for elucidating the molecular mechanisms underlying these diseases and for performing structure-based drug design. Although structural data are restricted to only a handful of GPCRs, homology models can be used as a proxy for those receptors not having crystal structures. However, many researchers working on GPCRs are not experienced homology modellers and are therefore unable to benefit from the information that can be gleaned from such three-dimensional models. Here, we present a comprehensive database called the GPCR-SSFE, which provides initial homology models of the transmembrane helices for a large variety of family A GPCRs. Extending on our previous theoretical work, we have developed an automated pipeline for GPCR homology modelling and applied it to a large set of family A GPCR sequences. Our pipeline is a fragment-based approach that exploits available family A crystal structures. The GPCR-SSFE database stores the template predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs. Users are able to browse the GPCR dataset according to their pharmacological classification or search for results using a UniProt entry name. It is also possible for a user to submit a GPCR sequence that is not contained in the database for analysis and homology model building. The models can be viewed using a Jmol applet and are also available for download along with the alignments. The data provided by GPCR-SSFE are useful for investigating general and detailed sequence-structure-function relationships

  12. Exploration of freely available web-interfaces for comparative homology modelling of microbial proteins.

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    Nema, Vijay; Pal, Sudhir Kumar

    2013-01-01

    This study was conducted to find the best suited freely available software for modelling of proteins by taking a few sample proteins. The proteins used were small to big in size with available crystal structures for the purpose of benchmarking. Key players like Phyre2, Swiss-Model, CPHmodels-3.0, Homer, (PS)2, (PS)(2)-V(2), Modweb were used for the comparison and model generation. Benchmarking process was done for four proteins, Icl, InhA, and KatG of Mycobacterium tuberculosis and RpoB of Thermus Thermophilus to get the most suited software. Parameters compared during analysis gave relatively better values for Phyre2 and Swiss-Model. This comparative study gave the information that Phyre2 and Swiss-Model make good models of small and large proteins as compared to other screened software. Other software was also good but is often not very efficient in providing full-length and properly folded structure.

  13. In silico sequence analysis and homology modeling of predicted beta-amylase 7-like protein in Brachypodium distachyon L.

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    ERTUĞRUL FILIZ

    2014-04-01

    Full Text Available Beta-amylase (β-amylase, EC 3.2.1.2 is an enzyme that catalyses hydrolysis of glucosidic bonds in polysaccharides. In this study, we analyzed protein sequence of predicted beta-amylase 7-like protein in Brachypodium distachyon. pI (isoelectric point value was found as 5.23 in acidic character, while the instability index (II was found as 50.28 with accepted unstable protein. The prediction of subcellular localization was revealed that the protein may reside in chloroplast by using CELLO v.2.5. The 3D structure of protein was performed using comparative homology modeling with SWISS-MODEL. The accuracy of the predicted 3D structure was checked using Ramachandran plot analysis showed that 95.4% in favored region. The results of our study contribute to understanding of β-amylase protein structure in grass species and will be scientific base for 3D modeling of beta-amylase proteins in further studies.

  14. Defining the limits of homology modeling in information-driven protein docking

    NARCIS (Netherlands)

    Garcia Lopes Maia Rodrigues, João; Melquiond, A S J; Karaca, E; Trellet, M; van Dijk, M; van Zundert, G C P; Schmitz, C; de Vries, S J; Bordogna, A; Bonati, L; Kastritis, P L; Bonvin, Alexandre M J J; Garcia Lopes Maia Rodrigues, João

    2013-01-01

    Information-driven docking is currently one of the most successful approaches to obtain structural models of protein interactions as demonstrated in the latest round of CAPRI. While various experimental and computational techniques can be used to retrieve information about the binding mode, the

  15. Homology modeling and virtual screening to discover potent inhibitors targeting the imidazole glycerophosphate dehydratase protein in Staphylococcus xylosus

    Science.gov (United States)

    Chen, Xing-Ru; Wang, Xiao-Ting; Hao, Mei-Qi; Zhou, Yong-Hui; Cui, Wen-Qiang; Xing, Xiao-Xu; Xu, Chang-Geng; Bai, Jing-Wen; Li, Yan-Hua

    2017-11-01

    The imidazole glycerophosphate dehydratase (IGPD) protein is a therapeutic target for herbicide discovery. It is also regarded as a possible target in Staphylococcus xylosus (S. xylosus) for solving mastitis in the dairy cow. The 3D structure of IGPD protein is essential for discovering novel inhibitors during high-throughput virtual screening. However, to date, the 3D structure of IGPD protein of S. xylosus has not been solved. In this study, a series of computational techniques including homology modeling, Ramachandran Plots, and Verify 3D were performed in order to construct an appropriate 3D model of IGPD protein of S. xylosus. Nine hits were identified from 2500 compounds by docking studies. Then, these 9 compounds were first tested in vitro in S. xylosus biofilm formation using crystal violet staining. One of the potential compounds, baicalin was shown to significantly inhibit S. xylosus biofilm formation. Finally, the baicalin was further evaluated, which showed better inhibition of biofilm formation capability in S. xylosus by scanning electron microscopy. Hence, we have predicted the structure of IGPD protein of S. xylosus using computational techniques. We further discovered the IGPD protein was targeted by baicalin compound which inhibited the biofilm formation in S. xylosus. Our findings here would provide implications for the further development of novel IGPD inhibitors for the treatment of dairy mastitis.

  16. Investigating homology between proteins using energetic profiles.

    Science.gov (United States)

    Wrabl, James O; Hilser, Vincent J

    2010-03-26

    Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may

  17. Investigating homology between proteins using energetic profiles.

    Directory of Open Access Journals (Sweden)

    James O Wrabl

    2010-03-01

    Full Text Available Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved

  18. Discovery of Novel Inhibitors for Nek6 Protein through Homology Model Assisted Structure Based Virtual Screening and Molecular Docking Approaches

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    P. Srinivasan

    2014-01-01

    Full Text Available Nek6 is a member of the NIMA (never in mitosis, gene A-related serine/threonine kinase family that plays an important role in the initiation of mitotic cell cycle progression. This work is an attempt to emphasize the structural and functional relationship of Nek6 protein based on homology modeling and binding pocket analysis. The three-dimensional structure of Nek6 was constructed by molecular modeling studies and the best model was further assessed by PROCHECK, ProSA, and ERRAT plot in order to analyze the quality and consistency of generated model. The overall quality of computed model showed 87.4% amino acid residues under the favored region. A 3 ns molecular dynamics simulation confirmed that the structure was reliable and stable. Two lead compounds (Binding database ID: 15666, 18602 were retrieved through structure-based virtual screening and induced fit docking approaches as novel Nek6 inhibitors. Hence, we concluded that the potential compounds may act as new leads for Nek6 inhibitors designing.

  19. Insight into the intermolecular recognition mechanism between Keap1 and IKKβ combining homology modelling, protein-protein docking, molecular dynamics simulations and virtual alanine mutation.

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    Zheng-Yu Jiang

    Full Text Available Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1, a substrate adaptor component of the Cullin3 (Cul3-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2 and IκB kinase β (IKKβ, which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI, the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.

  20. Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2.

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    Natarajaseenivasan, Kalimuthusamy; Shanmughapriya, Santhanam; Velineni, Sridhar; Artiushin, Sergey C; Timoney, John F

    2011-10-01

    Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a protein. The superposition of the Ca traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component. Copyright © 2011 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

  1. Modeling Non-homologous End Joining

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    Li, Yongfeng

    2013-01-01

    Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several NHEJ proteins such as Ku, DNA-PKcs, XRCC4, Ligase IV and so on. Once DSBs are generated, Ku is first recruited to the DNA end, followed by other NHEJ proteins for DNA end processing and ligation. Because of the direct ligation of break ends without the need for a homologous template, NHEJ turns out to be an error-prone but efficient repair pathway. Some mechanisms have been proposed of how the efficiency of NHEJ repair is affected. The type of DNA damage is an important factor of NHEJ repair. For instance, the length of DNA fragment may determine the recruitment efficiency of NHEJ protein such as Ku [1], or the complexity of the DNA breaks [2] is accounted for the choice of NHEJ proteins and subpathway of NHEJ repair. On the other hand, the chromatin structure also plays a role of the accessibility of NHEJ protein to the DNA damage site. In this talk, some mathematical models of NHEJ, that consist of series of biochemical reactions complying with the laws of chemical reaction (e.g. mass action, etc.), will be introduced. By mathematical and numerical analysis and parameter estimation, the models are able to capture the qualitative biological features and show good agreement with experimental data. As conclusions, from the viewpoint of modeling, how the NHEJ proteins are recruited will be first discussed for connection between the classical sequential model [4] and recently proposed two-phase model [5]. Then how the NHEJ repair pathway is affected, by the length of DNA fragment [6], the complexity of DNA damage [7] and the chromatin structure [8], will be addressed

  2. Structural modelling and comparative analysis of homologous, analogous and specific proteins from Trypanosoma cruzi versus Homo sapiens: putative drug targets for chagas' disease treatment.

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    Capriles, Priscila V S Z; Guimarães, Ana C R; Otto, Thomas D; Miranda, Antonio B; Dardenne, Laurent E; Degrave, Wim M

    2010-10-29

    Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic infection that causes thousands of deaths every year in Latin America. Therapeutic options remain inefficient, demanding the search for new drugs and/or new molecular targets. Such efforts can focus on proteins that are specific to the parasite, but analogous enzymes and enzymes with a three-dimensional (3D) structure sufficiently different from the corresponding host proteins may represent equally interesting targets. In order to find these targets we used the workflows MHOLline and AnEnΠ obtaining 3D models from homologous, analogous and specific proteins of Trypanosoma cruzi versus Homo sapiens. We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of T. cruzi. In combination with comparative genome analysis to Homo sapiens, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite. In this work, we present a set of 397 enzyme models of T. cruzi that can constitute potential structure-based drug targets to be investigated for the development of new strategies to fight Chagas' disease. The strategies presented here support the concept of structural analysis in conjunction with protein functional analysis as an interesting computational methodology to detect potential targets for structure-based rational drug design. For example, 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and triacylglycerol lipase (EC 3.1.1.3), classified as analogous proteins in relation to H. sapiens enzymes, were identified as new potential molecular targets.

  3. Clustering evolving proteins into homologous families.

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    Chan, Cheong Xin; Mahbob, Maisarah; Ragan, Mark A

    2013-04-08

    Clustering sequences into groups of putative homologs (families) is a critical first step in many areas of comparative biology and bioinformatics. The performance of clustering approaches in delineating biologically meaningful families depends strongly on characteristics of the data, including content bias and degree of divergence. New, highly scalable methods have recently been introduced to cluster the very large datasets being generated by next-generation sequencing technologies. However, there has been little systematic investigation of how characteristics of the data impact the performance of these approaches. Using clusters from a manually curated dataset as reference, we examined the performance of a widely used graph-based Markov clustering algorithm (MCL) and a greedy heuristic approach (UCLUST) in delineating protein families coded by three sets of bacterial genomes of different G+C content. Both MCL and UCLUST generated clusters that are comparable to the reference sets at specific parameter settings, although UCLUST tends to under-cluster compositionally biased sequences (G+C content 33% and 66%). Using simulated data, we sought to assess the individual effects of sequence divergence, rate heterogeneity, and underlying G+C content. Performance decreased with increasing sequence divergence, decreasing among-site rate variation, and increasing G+C bias. Two MCL-based methods recovered the simulated families more accurately than did UCLUST. MCL using local alignment distances is more robust across the investigated range of sequence features than are greedy heuristics using distances based on global alignment. Our results demonstrate that sequence divergence, rate heterogeneity and content bias can individually and in combination affect the accuracy with which MCL and UCLUST can recover homologous protein families. For application to data that are more divergent, and exhibit higher among-site rate variation and/or content bias, MCL may often be the better

  4. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model.

    Directory of Open Access Journals (Sweden)

    Jyoti Gupta

    Full Text Available We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo of Brugia malayi (B. malayi in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+ and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32 against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23 and pcD-Myo (41.6%±2.45. In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC to B. malayi infective larvae (L3. pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ and anti-inflammatory (IL-4, IL-10 cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of

  5. The Protein Model Portal

    OpenAIRE

    Arnold, Konstantin; Kiefer, Florian; Kopp, J?rgen; Battey, James N. D.; Podvinec, Michael; Westbrook, John D.; Berman, Helen M.; Bordoli, Lorenza; Schwede, Torsten

    2008-01-01

    Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploratio...

  6. Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

    International Nuclear Information System (INIS)

    Morton, Craig J.; Cameron, Rachel; Lawrence, Lynne J.; Lin Bo; Lowe, Melinda; Luttick, Angela; Mason, Anthony; McKimm-Breschkin, Jenny; Parker, Michael W.; Ryan, Jane; Smout, Michael; Sullivan, Jayne; Tucker, Simon P.; Young, Paul R.

    2003-01-01

    Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors

  7. Detection of the antiviral activity of epicatechin isolated from Salacia crassifolia (Celastraceae) against Mayaro virus based on protein C homology modelling and virtual screening.

    Science.gov (United States)

    Ferreira, P G; Ferraz, A C; Figueiredo, J E; Lima, C F; Rodrigues, V G; Taranto, A G; Ferreira, J M S; Brandão, G C; Vieira-Filho, S A; Duarte, L P; de Brito Magalhães, C L; de Magalhães, J C

    2018-02-24

    Mayaro fever, caused by Mayaro virus (MAYV) is a sub-lethal disease with symptoms that are easily confused with those of dengue fever, except for polyarthralgia, which may culminate in physical incapacitation. Recently, outbreaks of MAYV have been documented in metropolitan areas, and to date, there is no therapy or vaccine available. Moreover, there is no information regarding the three-dimensional structure of the viral proteins of MAYV, which is important in the search for antivirals. In this work, we constructed a three-dimensional model of protein C of MAYV by homology modelling, and this was employed in a manner similar to that of receptors in virtual screening studies to evaluate 590 molecules as prospective antiviral agents. In vitro bioassays were utilized to confirm the potential antiviral activity of the flavonoid epicatechin isolated from Salacia crassifolia (Celastraceae). The virtual screening showed that six flavonoids were promising ligands for protein C. The bioassays showed potent antiviral action of epicatechin, which protected the cells from almost all of the effects of viral infection. An effective concentration (EC 50 ) of 0.247 μmol/mL was observed with a selectivity index (SI) of 7. The cytotoxicity assay showed that epicatechin has low toxicity, with a 50% cytotoxic concentration (CC 50 ) greater than 1.723 µmol/mL. Epicatechin was found to be twice as potent as the reference antiviral ribavirin. Furthermore, a replication kinetics assay showed a strong inhibitory effect of epicatechin on MAYV growth, with a reduction of at least four logs in virus production. Our results indicate that epicatechin is a promising candidate for further testing as an antiviral agent against Mayaro virus and other alphaviruses.

  8. Refinement of homology-based protein structures by molecular dynamics simulation techniques

    NARCIS (Netherlands)

    Fan, H; Mark, AE

    The use of classical molecular dynamics simulations, performed in explicit water, for the refinement of structural models of proteins generated ab initio or based on homology has been investigated. The study involved a test set of 15 proteins that were previously used by Baker and coworkers to

  9. FastBLAST: homology relationships for millions of proteins.

    Directory of Open Access Journals (Sweden)

    Morgan N Price

    Full Text Available BACKGROUND: All-versus-all BLAST, which searches for homologous pairs of sequences in a database of proteins, is used to identify potential orthologs, to find new protein families, and to provide rapid access to these homology relationships. As DNA sequencing accelerates and data sets grow, all-versus-all BLAST has become computationally demanding. METHODOLOGY/PRINCIPAL FINDINGS: We present FastBLAST, a heuristic replacement for all-versus-all BLAST that relies on alignments of proteins to known families, obtained from tools such as PSI-BLAST and HMMer. FastBLAST avoids most of the work of all-versus-all BLAST by taking advantage of these alignments and by clustering similar sequences. FastBLAST runs in two stages: the first stage identifies additional families and aligns them, and the second stage quickly identifies the homologs of a query sequence, based on the alignments of the families, before generating pairwise alignments. On 6.53 million proteins from the non-redundant Genbank database ("NR", FastBLAST identifies new families 25 times faster than all-versus-all BLAST. Once the first stage is completed, FastBLAST identifies homologs for the average query in less than 5 seconds (8.6 times faster than BLAST and gives nearly identical results. For hits above 70 bits, FastBLAST identifies 98% of the top 3,250 hits per query. CONCLUSIONS/SIGNIFICANCE: FastBLAST enables research groups that do not have supercomputers to analyze large protein sequence data sets. FastBLAST is open source software and is available at http://microbesonline.org/fastblast.

  10. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    Directory of Open Access Journals (Sweden)

    Marc Lenoir

    2015-10-01

    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  11. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  12. Identification of putative agouti-related protein(87-132)-melanocortin-4 receptor interactions by homology molecular modeling and validation using chimeric peptide ligands.

    Science.gov (United States)

    Wilczynski, Andrzej; Wang, Xiang S; Joseph, Christine G; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Sorensen, Nicholas B; Shaw, Amanda M; Millard, William J; Richards, Nigel G; Haskell-Luevano, Carrie

    2004-04-22

    Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide

  13. MetaGO: Predicting Gene Ontology of Non-homologous Proteins Through Low-Resolution Protein Structure Prediction and Protein-Protein Network Mapping.

    Science.gov (United States)

    Zhang, Chengxin; Zheng, Wei; Freddolino, Peter L; Zhang, Yang

    2018-03-10

    Homology-based transferal remains the major approach to computational protein function annotations, but it becomes increasingly unreliable when the sequence identity between query and template decreases below 30%. We propose a novel pipeline, MetaGO, to deduce Gene Ontology attributes of proteins by combining sequence homology-based annotation with low-resolution structure prediction and comparison, and partner's homology-based protein-protein network mapping. The pipeline was tested on a large-scale set of 1000 non-redundant proteins from the CAFA3 experiment. Under the stringent benchmark conditions where templates with >30% sequence identity to the query are excluded, MetaGO achieves average F-measures of 0.487, 0.408, and 0.598, for Molecular Function, Biological Process, and Cellular Component, respectively, which are significantly higher than those achieved by other state-of-the-art function annotations methods. Detailed data analysis shows that the major advantage of the MetaGO lies in the new functional homolog detections from partner's homology-based network mapping and structure-based local and global structure alignments, the confidence scores of which can be optimally combined through logistic regression. These data demonstrate the power of using a hybrid model incorporating protein structure and interaction networks to deduce new functional insights beyond traditional sequence homology-based referrals, especially for proteins that lack homologous function templates. The MetaGO pipeline is available at http://zhanglab.ccmb.med.umich.edu/MetaGO/. Copyright © 2018. Published by Elsevier Ltd.

  14. Prefiltering Model for Homology Detection Algorithms on GPU.

    Science.gov (United States)

    Retamosa, Germán; de Pedro, Luis; González, Ivan; Tamames, Javier

    2016-01-01

    Homology detection has evolved over the time from heavy algorithms based on dynamic programming approaches to lightweight alternatives based on different heuristic models. However, the main problem with these algorithms is that they use complex statistical models, which makes it difficult to achieve a relevant speedup and find exact matches with the original results. Thus, their acceleration is essential. The aim of this article was to prefilter a sequence database. To make this work, we have implemented a groundbreaking heuristic model based on NVIDIA's graphics processing units (GPUs) and multicore processors. Depending on the sensitivity settings, this makes it possible to quickly reduce the sequence database by factors between 50% and 95%, while rejecting no significant sequences. Furthermore, this prefiltering application can be used together with multiple homology detection algorithms as a part of a next-generation sequencing system. Extensive performance and accuracy tests have been carried out in the Spanish National Centre for Biotechnology (NCB). The results show that GPU hardware can accelerate the execution times of former homology detection applications, such as National Centre for Biotechnology Information (NCBI), Basic Local Alignment Search Tool for Proteins (BLASTP), up to a factor of 4.

  15. Modeling Human Serum Albumin Tertiary Structure to Teach Upper-Division Chemistry Students Bioinformatics and Homology Modeling Basics

    Science.gov (United States)

    Petrovic, Dus?an; Zlatovic´, Mario

    2015-01-01

    A homology modeling laboratory experiment has been developed for an introductory molecular modeling course for upper-division undergraduate chemistry students. With this experiment, students gain practical experience in homology model preparation and assessment as well as in protein visualization using the educational version of PyMOL…

  16. A geometric model for Hochschild homology of Soergel bimodules

    DEFF Research Database (Denmark)

    Webster, Ben; Williamson, Geordie

    2008-01-01

    An important step in the calculation of the triply graded link homology of Khovanov and Rozansky is the determination of the Hochschild homology of Soergel bimodules for SL(n). We present a geometric model for this Hochschild homology for any simple group G, as B–equivariant intersection cohomology...... on generators whose degree is explicitly determined by the geometry of the orbit closure, and to describe its Hilbert series, proving a conjecture of Jacob Rasmussen....

  17. HomPPI: a class of sequence homology based protein-protein interface prediction methods

    Directory of Open Access Journals (Sweden)

    Dobbs Drena

    2011-06-01

    Full Text Available Abstract Background Although homology-based methods are among the most widely used methods for predicting the structure and function of proteins, the question as to whether interface sequence conservation can be effectively exploited in predicting protein-protein interfaces has been a subject of debate. Results We studied more than 300,000 pair-wise alignments of protein sequences from structurally characterized protein complexes, including both obligate and transient complexes. We identified sequence similarity criteria required for accurate homology-based inference of interface residues in a query protein sequence. Based on these analyses, we developed HomPPI, a class of sequence homology-based methods for predicting protein-protein interface residues. We present two variants of HomPPI: (i NPS-HomPPI (Non partner-specific HomPPI, which can be used to predict interface residues of a query protein in the absence of knowledge of the interaction partner; and (ii PS-HomPPI (Partner-specific HomPPI, which can be used to predict the interface residues of a query protein with a specific target protein. Our experiments on a benchmark dataset of obligate homodimeric complexes show that NPS-HomPPI can reliably predict protein-protein interface residues in a given protein, with an average correlation coefficient (CC of 0.76, sensitivity of 0.83, and specificity of 0.78, when sequence homologs of the query protein can be reliably identified. NPS-HomPPI also reliably predicts the interface residues of intrinsically disordered proteins. Our experiments suggest that NPS-HomPPI is competitive with several state-of-the-art interface prediction servers including those that exploit the structure of the query proteins. The partner-specific classifier, PS-HomPPI can, on a large dataset of transient complexes, predict the interface residues of a query protein with a specific target, with a CC of 0.65, sensitivity of 0.69, and specificity of 0.70, when homologs of

  18. Energetic frustrations in protein folding at residue resolution: a homologous simulation study of Im9 proteins.

    Directory of Open Access Journals (Sweden)

    Yunxiang Sun

    Full Text Available Energetic frustration is becoming an important topic for understanding the mechanisms of protein folding, which is a long-standing big biological problem usually investigated by the free energy landscape theory. Despite the significant advances in probing the effects of folding frustrations on the overall features of protein folding pathways and folding intermediates, detailed characterizations of folding frustrations at an atomic or residue level are still lacking. In addition, how and to what extent folding frustrations interact with protein topology in determining folding mechanisms remains unclear. In this paper, we tried to understand energetic frustrations in the context of protein topology structures or native-contact networks by comparing the energetic frustrations of five homologous Im9 alpha-helix proteins that share very similar topology structures but have a single hydrophilic-to-hydrophobic mutual mutation. The folding simulations were performed using a coarse-grained Gō-like model, while non-native hydrophobic interactions were introduced as energetic frustrations using a Lennard-Jones potential function. Energetic frustrations were then examined at residue level based on φ-value analyses of the transition state ensemble structures and mapped back to native-contact networks. Our calculations show that energetic frustrations have highly heterogeneous influences on the folding of the four helices of the examined structures depending on the local environment of the frustration centers. Also, the closer the introduced frustration is to the center of the native-contact network, the larger the changes in the protein folding. Our findings add a new dimension to the understanding of protein folding the topology determination in that energetic frustrations works closely with native-contact networks to affect the protein folding.

  19. HOMOLOGY MODELING AND MOLECULAR DYNAMICS STUDY OF MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. M. tuberculosis urease (MTU is an attractive target for chemotherapeutic intervention in tuberculosis by designing new safe and efficient enzyme inhibitors. A prerequisite for designing such inhibitors is an understanding of urease's three-dimensional (3D structure organization. 3D structure of M. tuberculosis urease is unknown. When experimental three-dimensional structure of a protein is not known, homology modeling, the most commonly used computational structure prediction method, is the technique of choice. This paper aimed to build a 3D-structure of M. tuberculosis urease by homology modeling and to study its stability by molecular dynamics simulations. Materials and methods. To build MTU model, five high-resolution X-ray structures of bacterial ureases with three-subunit composition (2KAU, 5G4H, 4UBP, 4СEU, and 4EPB have been selected as templates. For each template five stochastic alignments were created and for each alignment, a three-dimensional model was built. Then, each model was energy minimized and the models were ranked by quality Z-score. The MTU model with highest quality estimation amongst 25 potential models was selected. To further improve structure quality the model was refined by short molecular dynamics simulation that resulted in 20 snapshots which were rated according to their energy and the quality Z-score. The best scoring model having minimum energy was chosen as a final homology model of 3D structure for M. tuberculosis. The final model of MTU was also validated by using PDBsum and QMEAN servers. These checks confirmed good quality of MTU homology model. Results and discussion. Homology model of MTU is a nonamer (homotrimer of heterotrimers, (αβγ3 consisting of 2349 residues. In MTU heterotrimer, sub-units α, β, and γ tightly interact with each other at a surface of approximately 3000 Å2. Sub-unit α contains the enzyme active site with two Ni atoms coordinated by amino acid residues His347, His

  20. CPHmodels-3.0--remote homology modeling using structure-guided sequence profiles

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole

    2010-01-01

    CPHmodels-3.0 is a web server predicting protein 3D structure by use of single template homology modeling. The server employs a hybrid of the scoring functions of CPHmodels-2.0 and a novel remote homology-modeling algorithm. A query sequence is first attempted modeled using the fast CPHmodels-2.......0 profile-profile scoring function suitable for close homology modeling. The new computational costly remote homology-modeling algorithm is only engaged provided that no suitable PDB template is identified in the initial search. CPHmodels-3.0 was benchmarked in the CASP8 competition and produced models.......3 A. These performance values place the CPHmodels-3.0 method in the group of high performing 3D prediction tools. Beside its accuracy, one of the important features of the method is its speed. For most queries, the response time of the server is...

  1. KIN17, XPC, DNA-PKCS and XRCC4 proteins in the cellular response to DNA damages. Relations between nucleotide excision repair and non-homologous end joining in a human syn-genic model

    International Nuclear Information System (INIS)

    Despras, Emmanuelle

    2006-01-01

    The response to genotoxic stress involves many cellular factors in a complex network of mechanisms that aim to preserve the genetic integrity of the organism. These mechanisms enclose the detection and repair of DNA lesions, the regulation of transcription and replication and, eventually, the setting of cell death. Among the nuclear proteins involved in this response, kin17 proteins are zinc-finger proteins conserved through evolution and activated by ultraviolet (UV) or ionizing radiations (IR). We showed that human kin17 protein (HSAkin17) is found in the cell under a soluble form and a form tightly anchored to nuclear structures. A fraction of HSAkin17 protein is directly associated with chromatin. HSAkin17 protein is recruited to nuclear structures 24 hours after treatment with various agents inducing DNA double-strand breaks (DSB) and/or replication forks blockage. Moreover, the reduction of total HSAkin17 protein level sensitizes RKO cells to IR. We also present evidence for the involvement of HSAkin17 protein in DNA replication. This hypothesis was further confirmed by the biochemical demonstration of its belonging to the replication complex. HSAkin17 protein could link DNA replication and DNA repair, a defect in the HSAkin17 pathway leading to an increased radiosensitivity. In a second part, we studied the interactions between two DNA repair mechanisms: nucleotide excision repair (NER) and non-homologous end joining (NHEJ). NER repairs a wide variety of lesions inducing a distortion of the DNA double helix including UV-induced pyrimidine dimers. NHEJ allows the repair of DSB by direct joining of DNA ends. We used a syn-genic model for DNA repair defects based on RNA interference developed in the laboratory. Epstein-Barr virus-derived vectors (pEBV) allow long-term expression of siRNA and specific extinction of the targeted gene. The reduction of the expression of genes involved in NER (XPA and XPC) or NHEJ (DNA-PKcs and XRCC4) leads to the expected

  2. Interaction and dynamics of homologous pairing protein 2 (HOP2) and DNA studied by MD simulation

    Science.gov (United States)

    Moktan, Hem; Pezza, Roberto; Zhou, Donghua

    2015-03-01

    The homologous pairing protein 2 (Hop2) plays an important role in meiosis and DNA repair. Together with protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. We recently determined the NMR structure of the N-terminal domain of Hop2 and proposed a model of Protein-DNA complex based on NMR chemical shift perturbations and mutagenesis studies (Moktan, J Biol Chem 2014 10.1074/jbc.M114.548180). However structure and dynamics of the complex have not been studied at the atomic level yet. Here, we used classical MD simulations to study the interactions between the N-terminal HOP2 and DNA. The simulated results indicate that helix3 (H3) interacts with DNA in major groove and wing1 (W1) interacts mostly in minor groove mainly via direct hydrogen bonds. Also it is found that binding leads to reduced fluctuations in both protein and DNA. Several water bridge interactions have been identified. The residue-wise contributions to the interaction energy were evaluated. Also the functional motion of the protein is analyzed using principal component analysis. The results confirmed the importance of H3 and W1 for the stability of the complex, which is consistent with our previous experimental studies.

  3. The Interaction Pattern between a Homology Model of 40S Ribosomal S9 Protein of Rhizoctonia solani and 1-Hydroxyphenaize by Docking Study

    Directory of Open Access Journals (Sweden)

    Seema Dharni

    2014-01-01

    Full Text Available 1-Hydroxyphenazine (1-OH-PHZ, a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of 40S ribosomal S9 protein of R. solani that may be a promising approach for the management of fungal diseases.

  4. Regulation of homologous recombination repair protein Rad51 by Ku70

    International Nuclear Information System (INIS)

    Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

    2013-01-01

    Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

  5. Evolution of pH buffers and water homeostasis in eukaryotes: homology between humans and Acanthamoeba proteins.

    Science.gov (United States)

    Baig, Abdul M; Zohaib, R; Tariq, S; Ahmad, H R

    2018-02-01

    This study intended to trace the evolution of acid-base buffers and water homeostasis in eukaryotes. Acanthamoeba castellanii  was selected as a model unicellular eukaryote for this purpose. Homologies of proteins involved in pH and water regulatory mechanisms at cellular levels were compared between humans and A. castellanii. Amino acid sequence homology, structural homology, 3D modeling and docking prediction were done to show the extent of similarities between carbonic anhydrase 1 (CA1), aquaporin (AQP), band-3 protein and H + pump. Experimental assays were done with acetazolamide (AZM), brinzolamide and mannitol to observe their effects on the trophozoites of  A. castellanii.  The human CA1, AQP, band-3 protein and H + -transport proteins revealed similar proteins in Acanthamoeba. Docking showed the binding of AZM on amoebal AQP-like proteins.  Acanthamoeba showed transient shape changes and encystation at differential doses of brinzolamide, mannitol and AZM.  Conclusion: Water and pH regulating adapter proteins in Acanthamoeba and humans show significant homology, these mechanisms evolved early in the primitive unicellular eukaryotes and have remained conserved in multicellular eukaryotes.

  6. Density parameter estimation for finding clusters of homologous proteins-tracing actinobacterial pathogenicity lifestyles

    DEFF Research Database (Denmark)

    Röttger, Richard; Kalaghatgi, Prabhav; Sun, Peng

    2013-01-01

    Homology detection is a long-standing challenge in computational biology. To tackle this problem, typically all-versus-all BLAST results are coupled with data partitioning approaches resulting in clusters of putative homologous proteins. One of the main problems, however, has been widely neglecte...

  7. The Protein Model Portal.

    Science.gov (United States)

    Arnold, Konstantin; Kiefer, Florian; Kopp, Jürgen; Battey, James N D; Podvinec, Michael; Westbrook, John D; Berman, Helen M; Bordoli, Lorenza; Schwede, Torsten

    2009-03-01

    Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploration of the protein structure space. One of the challenges in using model information effectively has been to access all models available for a specific protein in heterogeneous formats at different sites using various incompatible accession code systems. Often, structure models for hundreds of proteins can be derived from a given experimentally determined structure, using a variety of established methods. This has been done by all of the PSI centers, and by various independent modeling groups. The goal of the Protein Model Portal (PMP) is to provide a single portal which gives access to the various models that can be leveraged from PSI targets and other experimental protein structures. A single interface allows all existing pre-computed models across these various sites to be queried simultaneously, and provides links to interactive services for template selection, target-template alignment, model building, and quality assessment. The current release of the portal consists of 7.6 million model structures provided by different partner resources (CSMP, JCSG, MCSG, NESG, NYSGXRC, JCMM, ModBase, SWISS-MODEL Repository). The PMP is available at http://www.proteinmodelportal.org and from the PSI Structural Genomics Knowledgebase.

  8. Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

    Science.gov (United States)

    Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

    2014-02-01

    The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

  9. Structural and Sequence Similarities of Hydra Xeroderma Pigmentosum A Protein to Human Homolog Suggest Early Evolution and Conservation

    Directory of Open Access Journals (Sweden)

    Apurva Barve

    2013-01-01

    Full Text Available Xeroderma pigmentosum group A (XPA is a protein that binds to damaged DNA, verifies presence of a lesion, and recruits other proteins of the nucleotide excision repair (NER pathway to the site. Though its homologs from yeast, Drosophila, humans, and so forth are well studied, XPA has not so far been reported from protozoa and lower animal phyla. Hydra is a fresh-water cnidarian with a remarkable capacity for regeneration and apparent lack of organismal ageing. Cnidarians are among the first metazoa with a defined body axis, tissue grade organisation, and nervous system. We report here for the first time presence of XPA gene in hydra. Putative protein sequence of hydra XPA contains nuclear localization signal and bears the zinc-finger motif. It contains two conserved Pfam domains and various characterized features of XPA proteins like regions for binding to excision repair cross-complementing protein-1 (ERCC1 and replication protein A 70 kDa subunit (RPA70 proteins. Hydra XPA shows a high degree of similarity with vertebrate homologs and clusters with deuterostomes in phylogenetic analysis. Homology modelling corroborates the very close similarity between hydra and human XPA. The protein thus most likely functions in hydra in the same manner as in other animals, indicating that it arose early in evolution and has been conserved across animal phyla.

  10. CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction

    KAUST Repository

    Cui, Xuefeng; Lu, Zhiwu; Wang, Sheng; Jing-Yan Wang, Jim; Gao, Xin

    2016-01-01

    Motivation: Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment

  11. CPHmodels-3.0--remote homology modeling using structure-guided sequence profiles.

    Science.gov (United States)

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole; Petersen, Thomas Nordahl

    2010-07-01

    CPHmodels-3.0 is a web server predicting protein 3D structure by use of single template homology modeling. The server employs a hybrid of the scoring functions of CPHmodels-2.0 and a novel remote homology-modeling algorithm. A query sequence is first attempted modeled using the fast CPHmodels-2.0 profile-profile scoring function suitable for close homology modeling. The new computational costly remote homology-modeling algorithm is only engaged provided that no suitable PDB template is identified in the initial search. CPHmodels-3.0 was benchmarked in the CASP8 competition and produced models for 94% of the targets (117 out of 128), 74% were predicted as high reliability models (87 out of 117). These achieved an average RMSD of 4.6 A when superimposed to the 3D structure. The remaining 26% low reliably models (30 out of 117) could superimpose to the true 3D structure with an average RMSD of 9.3 A. These performance values place the CPHmodels-3.0 method in the group of high performing 3D prediction tools. Beside its accuracy, one of the important features of the method is its speed. For most queries, the response time of the server is web server is available at http://www.cbs.dtu.dk/services/CPHmodels/.

  12. Isolation and identification of the human homolog of a new p53-binding protein, Mdmx

    NARCIS (Netherlands)

    Shvarts, A.; Bazuine, M.; Dekker, P.; Ramos, Y. F.; Steegenga, W. T.; Merckx, G.; van Ham, R. C.; van der Houven van Oordt, W.; van der Eb, A. J.; Jochemsen, A. G.

    1997-01-01

    We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a

  13. Automated Protein Structure Modeling with SWISS-MODEL Workspace and the Protein Model Portal

    OpenAIRE

    Bordoli, Lorenza; Schwede, Torsten

    2012-01-01

    Comparative protein structure modeling is a computational approach to build three-dimensional structural models for proteins using experimental structures of related protein family members as templates. Regular blind assessments of modeling accuracy have demonstrated that comparative protein structure modeling is currently the most reliable technique to model protein structures. Homology models are often sufficiently accurate to substitute for experimental structures in a wide variety of appl...

  14. An Approach for Zika Virus Inhibition Using Homology Structure of the Envelope Protein

    Czech Academy of Sciences Publication Activity Database

    Fernando, S.; Fernando, T.; Štefánik, M.; Eyer, Luděk; Růžek, Daniel

    2016-01-01

    Roč. 58, č. 12 (2016), s. 801-806 ISSN 1073-6085 Institutional support: RVO:60077344 Keywords : Zika virus * homology model * druggability * drug discovery Subject RIV: EE - Microbiology, Virology Impact factor: 1.634, year: 2016

  15. Uncoupling protein homologs may provide a link between mitochondria, metabolism and lifespan.

    Science.gov (United States)

    Wolkow, Catherine A; Iser, Wendy B

    2006-05-01

    Uncoupling proteins (UCPs), which dissipate the mitochondrial proton gradient, have the ability to decouple mitochodrial respiration from ATP production. Since mitochondrial electron transport is a major source of free radical production, it is possible that UCP activity might impact free radical production. Free radicals can react with and damage cellular proteins, DNA and lipids. Accumulated damage from oxidative stress is believed to be a major contributor to cellular decline during aging. If UCP function were to impact mitochondrial free radical production, then one would expect to find a link between UCP activity and aging. This theory has recently been tested in a handful of organisms whose genomes contain UCP1 homologs. Interestingly, these experiments indicate that UCP homologs can affect lifespan, although they do not support a simple relationship between UCP activity and aging. Instead, UCP-like proteins appear to have a variety of effects on lifespan, and on pathways implicated in lifespan regulation. One possible explanation for this complex picture is that UCP homologs may have tissue-specific effects that complicate their effects on aging. Furthermore, the functional analysis of UCP1 homologs is incomplete. Thus, these proteins may perform functions in addition to, or instead of, mitochondrial uncoupling. Although these studies have not revealed a clear picture of UCP effects on aging, they have contributed to the growing knowledge base for these interesting proteins. Future biochemical and genetic investigation of UCP-like proteins will do much to clarify their functions and to identify the regulatory networks in which they are involved.

  16. Comprehensive Analysis of Homologous Proteins for Specific Drug ...

    African Journals Online (AJOL)

    ... minimize drug failures by predicting drug efficacy and toxicity. One of the most important pathogenic bacterium is Aeromonas species which causes tissue damage, acute gastroenteritis and neonatal septicemia. Bacterial proteins are the ultimate target to inhibit their growth and these are the executors of cellular function.

  17. Functional Coverage of the Human Genome by Existing Structures, Structural Genomics Targets, and Homology Models.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available The bias in protein structure and function space resulting from experimental limitations and targeting of particular functional classes of proteins by structural biologists has long been recognized, but never continuously quantified. Using the Enzyme Commission and the Gene Ontology classifications as a reference frame, and integrating structure data from the Protein Data Bank (PDB, target sequences from the structural genomics projects, structure homology derived from the SUPERFAMILY database, and genome annotations from Ensembl and NCBI, we provide a quantified view, both at the domain and whole-protein levels, of the current and projected coverage of protein structure and function space relative to the human genome. Protein structures currently provide at least one domain that covers 37% of the functional classes identified in the genome; whole structure coverage exists for 25% of the genome. If all the structural genomics targets were solved (twice the current number of structures in the PDB, it is estimated that structures of one domain would cover 69% of the functional classes identified and complete structure coverage would be 44%. Homology models from existing experimental structures extend the 37% coverage to 56% of the genome as single domains and 25% to 31% for complete structures. Coverage from homology models is not evenly distributed by protein family, reflecting differing degrees of sequence and structure divergence within families. While these data provide coverage, conversely, they also systematically highlight functional classes of proteins for which structures should be determined. Current key functional families without structure representation are highlighted here; updated information on the "most wanted list" that should be solved is available on a weekly basis from http://function.rcsb.org:8080/pdb/function_distribution/index.html.

  18. Metabolism of homologous and heterologous serum proteins in garter snakes (Thamnophis ordinoides)

    International Nuclear Information System (INIS)

    Leong, D.; Coe, J.E.

    1978-01-01

    The half-life (Tsub(1/2) of serum immunoglobulin (Ig) and albumin from snakes and mammals were determined in both garter snakes (Thamnophis ordinoides) and mice (Mus musculus). Metabolism of serum proteins in snakes was similar to mammalian protein metabolism in that homologous serum albumin had shorter Tsub(1/2) (16 days) than IgG (38 days). Also, reptilian and mammalian serum proteins had a relatively longer Tsub(1/2) when injected into closely related species. Thus mammalian serum Ig (rabbit gamma globulin (RGG)) had a shorter Tsub(1/2) (6.3 days) in snake than did homologous snake IgG (38 days), whereas in mice, RGG had a longer Tsub(1/2) (3.8 days) than snake Ig (0.9 days). Differences between metabolism of homologous and heterologous albumins were apparent only in snakes in which the Tsub(1/2) of homologous albumin was approximately 8-fold greater than mammalian albumin. These results indicate that metabolism of both Ig and albumin in snakes is regulated by specific receptors whereas albumin receptors have been difficult to demonstrate in mammals. The results of this study suggest that one of the factors determining the metabolism of a protein is its foreignness to the host perhaps because of receptor cross reactions. (author)

  19. Protein remote homology detection based on bidirectional long short-term memory.

    Science.gov (United States)

    Li, Shumin; Chen, Junjie; Liu, Bin

    2017-10-10

    Protein remote homology detection plays a vital role in studies of protein structures and functions. Almost all of the traditional machine leaning methods require fixed length features to represent the protein sequences. However, it is never an easy task to extract the discriminative features with limited knowledge of proteins. On the other hand, deep learning technique has demonstrated its advantage in automatically learning representations. It is worthwhile to explore the applications of deep learning techniques to the protein remote homology detection. In this study, we employ the Bidirectional Long Short-Term Memory (BLSTM) to learn effective features from pseudo proteins, also propose a predictor called ProDec-BLSTM: it includes input layer, bidirectional LSTM, time distributed dense layer and output layer. This neural network can automatically extract the discriminative features by using bidirectional LSTM and the time distributed dense layer. Experimental results on a widely-used benchmark dataset show that ProDec-BLSTM outperforms other related methods in terms of both the mean ROC and mean ROC50 scores. This promising result shows that ProDec-BLSTM is a useful tool for protein remote homology detection. Furthermore, the hidden patterns learnt by ProDec-BLSTM can be interpreted and visualized, and therefore, additional useful information can be obtained.

  20. Stringent homology-based prediction of H. sapiens-M. tuberculosis H37Rv protein-protein interactions.

    Science.gov (United States)

    Zhou, Hufeng; Gao, Shangzhi; Nguyen, Nam Ninh; Fan, Mengyuan; Jin, Jingjing; Liu, Bing; Zhao, Liang; Xiong, Geng; Tan, Min; Li, Shijun; Wong, Limsoon

    2014-04-08

    H. sapiens-M. tuberculosis H37Rv protein-protein interaction (PPI) data are essential for understanding the infection mechanism of the formidable pathogen M. tuberculosis H37Rv. Computational prediction is an important strategy to fill the gap in experimental H. sapiens-M. tuberculosis H37Rv PPI data. Homology-based prediction is frequently used in predicting both intra-species and inter-species PPIs. However, some limitations are not properly resolved in several published works that predict eukaryote-prokaryote inter-species PPIs using intra-species template PPIs. We develop a stringent homology-based prediction approach by taking into account (i) differences between eukaryotic and prokaryotic proteins and (ii) differences between inter-species and intra-species PPI interfaces. We compare our stringent homology-based approach to a conventional homology-based approach for predicting host-pathogen PPIs, based on cellular compartment distribution analysis, disease gene list enrichment analysis, pathway enrichment analysis and functional category enrichment analysis. These analyses support the validity of our prediction result, and clearly show that our approach has better performance in predicting H. sapiens-M. tuberculosis H37Rv PPIs. Using our stringent homology-based approach, we have predicted a set of highly plausible H. sapiens-M. tuberculosis H37Rv PPIs which might be useful for many of related studies. Based on our analysis of the H. sapiens-M. tuberculosis H37Rv PPI network predicted by our stringent homology-based approach, we have discovered several interesting properties which are reported here for the first time. We find that both host proteins and pathogen proteins involved in the host-pathogen PPIs tend to be hubs in their own intra-species PPI network. Also, both host and pathogen proteins involved in host-pathogen PPIs tend to have longer primary sequence, tend to have more domains, tend to be more hydrophilic, etc. And the protein domains from both

  1. Characterization of the Zebrafish Homolog of Zipper Interacting Protein Kinase

    Directory of Open Access Journals (Sweden)

    Brandon W. Carr

    2014-06-01

    Full Text Available Zipper-interacting protein kinase (ZIPK is a conserved vertebrate-specific regulator of actomyosin contractility in smooth muscle and non-muscle cells. Murine ZIPK has undergone an unusual divergence in sequence and regulation compared to other ZIPK orthologs. In humans, subcellular localization is controlled by phosphorylation of threonines 299 and 300. In contrast, ZIPK subcellular localization in mouse and rat is controlled by interaction with PAR-4. We carried out a comparative biochemical characterization of the regulation of the zebrafish ortholog of ZIPK. Like the human orthologs zebrafish ZIPK undergoes nucleocytoplasmic-shuttling and is abundant in the cytoplasm, unlike the primarily nuclear rat ZIPK. Rat ZIPK, but not human or zebrafish ZIPK, interacts with zebrafish PAR-4. Mutation of the conserved residues required for activation of the mammalian orthologs abrogated activity of the zebrafish ZIPK. In contrast to the human ortholog, mutation of threonine 299 and 300 in the zebrafish ZIPK has no effect on the activity or subcellular localization. Thus, we found that zebrafish ZIPK functions in a manner most similar to the human ZIPK and quite distinct from murine orthologs, yet the regulation of subcellular localization is not conserved.

  2. Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

    OpenAIRE

    Martin, M E; Piette, J; Yaniv, M; Tang, W J; Folk, W R

    1988-01-01

    The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription fac...

  3. Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Mate Suzanne E

    2012-09-01

    Full Text Available Abstract Background Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ. We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs. Methods Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle. Results Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle. Conclusion EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.

  4. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    Directory of Open Access Journals (Sweden)

    Michał Śmiga

    Full Text Available Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins and T. forsythia (Tfo protein and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

  5. Evolutionary distance from human homologs reflects allergenicity of animal food proteins.

    Science.gov (United States)

    Jenkins, John A; Breiteneder, Heimo; Mills, E N Clare

    2007-12-01

    In silico analysis of allergens can identify putative relationships among protein sequence, structure, and allergenic properties. Such systematic analysis reveals that most plant food allergens belong to a restricted number of protein superfamilies, with pollen allergens behaving similarly. We have investigated the structural relationships of animal food allergens and their evolutionary relatedness to human homologs to define how closely a protein must resemble a human counterpart to lose its allergenic potential. Profile-based sequence homology methods were used to classify animal food allergens into Pfam families, and in silico analyses of their evolutionary and structural relationships were performed. Animal food allergens could be classified into 3 main families--tropomyosins, EF-hand proteins, and caseins--along with 14 minor families each composed of 1 to 3 allergens. The evolutionary relationships of each of these allergen superfamilies showed that in general, proteins with a sequence identity to a human homolog above approximately 62% were rarely allergenic. Single substitutions in otherwise highly conserved regions containing IgE epitopes in EF-hand parvalbumins may modulate allergenicity. These data support the premise that certain protein structures are more allergenic than others. Contrasting with plant food allergens, animal allergens, such as the highly conserved tropomyosins, challenge the capability of the human immune system to discriminate between foreign and self-proteins. Such immune responses run close to becoming autoimmune responses. Exploiting the closeness between animal allergens and their human homologs in the development of recombinant allergens for immunotherapy will need to consider the potential for developing unanticipated autoimmune responses.

  6. Classical Bovine Spongiform Encephalopathy by Transmission of H-Type Prion in Homologous Prion Protein Context

    Science.gov (United States)

    Andréoletti, Olivier; Lacroux, Caroline; Prieto, Irene; Lorenzo, Patricia; Larska, Magdalena; Baron, Thierry; Espinosa, Juan-Carlos

    2011-01-01

    Bovine spongiform encephalopathy (BSE) and BSE-related disorders have been associated with a single major prion strain. Recently, 2 atypical, presumably sporadic forms of BSE have been associated with 2 distinct prion strains that are characterized mainly by distinct Western blot profiles of abnormal protease-resistant prion protein (PrPres), named high-type (BSE-H) and low-type (BSE-L), that also differed from classical BSE. We characterized 5 atypical BSE-H isolates by analyzing their molecular and neuropathologic properties during transmission in transgenic mice expressing homologous bovine prion protein. Unexpectedly, in several inoculated animals, strain features emerged that were highly similar to those of classical BSE agent. These findings demonstrate the capability of an atypical bovine prion to acquire classical BSE–like properties during propagation in a homologous bovine prion protein context and support the view that the epidemic BSE agent could have originated from such a cattle prion. PMID:21888788

  7. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  8. Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

    Science.gov (United States)

    Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

    2016-11-01

    Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is

  9. Determination and validation of mTOR kinase-domain 3D structure by homology modeling

    Directory of Open Access Journals (Sweden)

    Lakhlili W

    2015-07-01

    Full Text Available Wiame Lakhlili,1 Gwénaël Chevé,2 Abdelaziz Yasri,2 Azeddine Ibrahimi1 1Laboratoire de Biotechnologie (MedBiotech, Faculté de Médecine et de Pharmacie de Rabat, Université Mohammed V de Rabat, Rabat, Morroco; 2OriBase Pharma, Cap Gamma, Parc Euromédecine, Montpellier, France Abstract: The AKT/mammalian target of rapamycin (mTOR pathway is considered as one of the commonly activated and deregulated signaling pathways in human cancer. mTOR is associated with other proteins in two molecular complexes: mTOR complex 1/Raptor and the mTOR complex 2/Rictor. Using the crystal structure of the related lipid kinase PI3Kγ, we built a model of the catalytic region of mTOR. The modeling of the three-dimensional (3D structure of the mTOR was performed by homology modeling program SWISS-MODEL. The quality and validation of the obtained model were performed using PROCHECK and PROVE softwares. The overall stereochemical property of the protein was assessed by the Ramachandran plot. The model validation was also done by docking of known inhibitors. In this paper, we describe and validate a 3D model for the mTOR catalytic site.Keywords: mTOR, homology modeling, mTOR kinase-domain, docking

  10. CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction

    KAUST Repository

    Cui, Xuefeng

    2016-06-15

    Motivation: Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment, threading and alignment-free methods, protein homology detection remains a challenging open problem. Recently, network methods that try to find transitive paths in the protein structure space demonstrate the importance of incorporating network information of the structure space. Yet, current methods merge the sequence space and the structure space into a single space, and thus introduce inconsistency in combining different sources of information. Method: We present a novel network-based protein homology detection method, CMsearch, based on cross-modal learning. Instead of exploring a single network built from the mixture of sequence and structure space information, CMsearch builds two separate networks to represent the sequence space and the structure space. It then learns sequence–structure correlation by simultaneously taking sequence information, structure information, sequence space information and structure space information into consideration. Results: We tested CMsearch on two challenging tasks, protein homology detection and protein structure prediction, by querying all 8332 PDB40 proteins. Our results demonstrate that CMsearch is insensitive to the similarity metrics used to define the sequence and the structure spaces. By using HMM–HMM alignment as the sequence similarity metric, CMsearch clearly outperforms state-of-the-art homology detection methods and the CASP-winning template-based protein structure prediction methods.

  11. Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode.

    Science.gov (United States)

    Krishnamoorthy, Ezhilarasi; Hassan, Sameer; Hanna, Luke Elizabeth; Padmalayam, Indira; Rajaram, Rama; Viswanathan, Vijay

    2017-05-07

    Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. GPCRM: a homology modeling web service with triple membrane-fitted quality assessment of GPCR models.

    Science.gov (United States)

    Miszta, Przemyslaw; Pasznik, Pawel; Jakowiecki, Jakub; Sztyler, Agnieszka; Latek, Dorota; Filipek, Slawomir

    2018-05-21

    Due to the involvement of G protein-coupled receptors (GPCRs) in most of the physiological and pathological processes in humans they have been attracting a lot of attention from pharmaceutical industry as well as from scientific community. Therefore, the need for new, high quality structures of GPCRs is enormous. The updated homology modeling service GPCRM (http://gpcrm.biomodellab.eu/) meets those expectations by greatly reducing the execution time of submissions (from days to hours/minutes) with nearly the same average quality of obtained models. Additionally, due to three different scoring functions (Rosetta, Rosetta-MP, BCL::Score) it is possible to select accurate models for the required purposes: the structure of the binding site, the transmembrane domain or the overall shape of the receptor. Currently, no other web service for GPCR modeling provides this possibility. GPCRM is continually upgraded in a semi-automatic way and the number of template structures has increased from 20 in 2013 to over 90 including structures the same receptor with different ligands which can influence the structure not only in the on/off manner. Two types of protein viewers can be used for visual inspection of obtained models. The extended sortable tables with available templates provide links to external databases and display ligand-receptor interactions in visual form.

  13. Biochemical and Structural Analysis of Hormone-sensitive Lipase Homolog EstE7: Insight into the Stabilized Dimerization of HSL-Homolog Proteins

    International Nuclear Information System (INIS)

    Nam, Ki Hyun; Park, Sung Ha; Lee, Won Ho; Hwang, Kwang Yeon

    2010-01-01

    Hormone sensitive lipase (HSL) plays a major role in energy homeostasis and lipid metabolism. Several crystal structures of HSL-homolog proteins have been identified, which has led to a better understanding of its molecular function. HSLhomolog proteins exit as both monomer and dimer, but the biochemical and structural basis for such oligomeric states has not been successfully elucidated. Therefore, we determined the crystal structure of HSL-homolog protein EstE7 from a metagenome library at 2.2 A resolution and characterized the oligomeric states of EstE7 both structurally and biochemically. EstE7 protein prefers the dimeric state in solution, which is supported by its higher enzymatic activity in the dimeric state. In the crystal form, EstE7 protein shows two-types of dimeric interface. Specifically, dimerization via the external β8-strand occurred through tight association between two pseudosymmetric folds via salt bridges, hydrogen bonds and van der Waals interactions. This dimer formation was similar to that of other HSL-homolog protein structures such as AFEST, BEFA, and EstE1. We anticipate that our results will provide insight into the oligomeric state of HSLhomolog proteins

  14. The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation

    Directory of Open Access Journals (Sweden)

    Litscher Eveline S

    2006-04-01

    Full Text Available Abstract Background Hundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP domains. ZP domain proteins perform highly diverse functions, ranging from structural to receptorial, and mutations in their genes are responsible for a number of severe human diseases. Recently, PLAC1, Oosp1-3, Papillote and CG16798 proteins were identified that share sequence homology with the N-terminal half of the ZP domain (ZP-N, but not with its C-terminal half (ZP-C. The functional significance of this partial conservation is unknown. Results By exploiting a highly engineered bacterial strain, we expressed in soluble form the PLAC1-homology region of mammalian sperm receptor ZP3 as a fusion to maltose binding protein. Mass spectrometry showed that the 4 conserved Cys residues within the ZP-N moiety of the fusion protein adopt the same disulfide bond connectivity as in full-length native ZP3, indicating that it is correctly folded, and electron microscopy and biochemical analyses revealed that it assembles into filaments. Conclusion These findings provide a function for PLAC1-like proteins and, by showing that ZP-N is a biologically active folding unit, prompt a re-evaluation of the architecture of the ZP domain and its polymers. Furthermore, they suggest that ZP-C might play a regulatory role in the assembly of ZP domain protein complexes.

  15. The K Domain Mediates Homologous and Heterologous Interactions Between FLC and SVP Proteins of Brassica juncea

    Directory of Open Access Journals (Sweden)

    Ma Guanpeng

    2015-07-01

    Full Text Available The transcription factors FLOWERING LOCUS C (FLC and SHORT VEGETATIVE PHASE (SVP can interact to form homologous and heterologous protein complexes that regulate flowering time in Brassica juncea Coss. (Mustard.Previous studies showed that protein interactions were mediated by the K domain, which contains the subdomains K1, K2 and K3. However, it remains unknown how the subdomains mediate the interactions between FLC and SVP. In the present study, we constructed several mutants of subdomains K1–K3 and investigated the mechanisms involved in the heterologous interaction of BjFLC/BjSVP and in the homologous interaction of BjFLC/BjFLC or BjSVP/BjSVP. Yeast two-hybrid and β-Galactosidase activity assays showed that the 19 amino acids of the K1 subdomain in BjSVP and the 17 amino acids of the K1 subdomain in BjFLC were functional subdomains that interact with each other to mediate hetero-dimerization. The heterologous interaction was enhanced by the K2 subdomain of BjSVP protein, but weakened by its interhelical domain L2. The heterologous interaction was also enhanced by the K2 subdomain of BjFLC protein, but weakened by its K3 subdomain. The homologous interaction of BjSVP was mediated by the full K-domain. However, the homologous interaction of BjFLC was regulated only by its K1 and weakened by its K2 and K3 subdomains. The results provided new insights into the interactions between FLC and SVP, which will be valuable for further studies on the molecular regulation mechanisms of the regulation of flowering time in B. juncea and other Brassicaceae.

  16. Beauty is in the eye of the beholder: proteins can recognize binding sites of homologous proteins in more than one way.

    Directory of Open Access Journals (Sweden)

    Juliette Martin

    2010-06-01

    Full Text Available Understanding the mechanisms of protein-protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein-protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein-protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.

  17. MollDE: a homology modeling framework you can click with.

    Science.gov (United States)

    Canutescu, Adrian A; Dunbrack, Roland L

    2005-06-15

    Molecular Integrated Development Environment (MolIDE) is an integrated application designed to provide homology modeling tools and protocols under a uniform, user-friendly graphical interface. Its main purpose is to combine the most frequent modeling steps in a semi-automatic, interactive way, guiding the user from the target protein sequence to the final three-dimensional protein structure. The typical basic homology modeling process is composed of building sequence profiles of the target sequence family, secondary structure prediction, sequence alignment with PDB structures, assisted alignment editing, side-chain prediction and loop building. All of these steps are available through a graphical user interface. MolIDE's user-friendly and streamlined interactive modeling protocol allows the user to focus on the important modeling questions, hiding from the user the raw data generation and conversion steps. MolIDE was designed from the ground up as an open-source, cross-platform, extensible framework. This allows developers to integrate additional third-party programs to MolIDE. http://dunbrack.fccc.edu/molide/molide.php rl_dunbrack@fccc.edu.

  18. Homologous Basal Ganglia Network Models in Physiological and Parkinsonian Conditions

    Directory of Open Access Journals (Sweden)

    Jyotika Bahuguna

    2017-08-01

    Full Text Available The classical model of basal ganglia has been refined in recent years with discoveries of subpopulations within a nucleus and previously unknown projections. One such discovery is the presence of subpopulations of arkypallidal and prototypical neurons in external globus pallidus, which was previously considered to be a primarily homogeneous nucleus. Developing a computational model of these multiple interconnected nuclei is challenging, because the strengths of the connections are largely unknown. We therefore use a genetic algorithm to search for the unknown connectivity parameters in a firing rate model. We apply a binary cost function derived from empirical firing rate and phase relationship data for the physiological and Parkinsonian conditions. Our approach generates ensembles of over 1,000 configurations, or homologies, for each condition, with broad distributions for many of the parameter values and overlap between the two conditions. However, the resulting effective weights of connections from or to prototypical and arkypallidal neurons are consistent with the experimental data. We investigate the significance of the weight variability by manipulating the parameters individually and cumulatively, and conclude that the correlation observed between the parameters is necessary for generating the dynamics of the two conditions. We then investigate the response of the networks to a transient cortical stimulus, and demonstrate that networks classified as physiological effectively suppress activity in the internal globus pallidus, and are not susceptible to oscillations, whereas parkinsonian networks show the opposite tendency. Thus, we conclude that the rates and phase relationships observed in the globus pallidus are predictive of experimentally observed higher level dynamical features of the physiological and parkinsonian basal ganglia, and that the multiplicity of solutions generated by our method may well be indicative of a natural

  19. Automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal.

    Science.gov (United States)

    Bordoli, Lorenza; Schwede, Torsten

    2012-01-01

    Comparative protein structure modeling is a computational approach to build three-dimensional structural models for proteins using experimental structures of related protein family members as templates. Regular blind assessments of modeling accuracy have demonstrated that comparative protein structure modeling is currently the most reliable technique to model protein structures. Homology models are often sufficiently accurate to substitute for experimental structures in a wide variety of applications. Since the usefulness of a model for specific application is determined by its accuracy, model quality estimation is an essential component of protein structure prediction. Comparative protein modeling has become a routine approach in many areas of life science research since fully automated modeling systems allow also nonexperts to build reliable models. In this chapter, we describe practical approaches for automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal.

  20. Discovery of a Dipeptide Epimerase Enzymatic Function Guided by Homology Modeling and Virtual Screening

    Energy Technology Data Exchange (ETDEWEB)

    Kalyanaraman, C.; Imker, H; Fedorov, A; Fedorov, E; Glasner, M; Babbitt, P; Almo, S; Gerlt, J; Jacobson, M

    2008-01-01

    We have developed a computational approach to aid the assignment of enzymatic function for uncharacterized proteins that uses homology modeling to predict the structure of the binding site and in silico docking to identify potential substrates. We apply this method to proteins in the functionally diverse enolase superfamily that are homologous to the characterized L-Ala-D/L-Glu epimerase from Bacillus subtilis. In particular, a protein from Thermotoga martima was predicted to have different substrate specificity, which suggests that it has a different, but as yet unknown, biological function. This prediction was experimentally confirmed, resulting in the assignment of epimerase activity for L-Ala-D/L-Phe, L-Ala-D/L-Tyr, and L-Ala-D/L-His, whereas the enzyme is annotated incorrectly in GenBank as muconate cycloisomerase. Subsequently, crystal structures of the enzyme were determined in complex with three substrates, showing close agreement with the computational models and revealing the structural basis for the observed substrate selectivity.

  1. Limited tryptic proteolysis of the benzodiazepine binding proteins in different species reveals structural homologies.

    Science.gov (United States)

    Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J

    1988-12-01

    Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.

  2. Protein thermostability prediction within homologous families using temperature-dependent statistical potentials.

    Directory of Open Access Journals (Sweden)

    Fabrizio Pucci

    Full Text Available The ability to rationally modify targeted physical and biological features of a protein of interest holds promise in numerous academic and industrial applications and paves the way towards de novo protein design. In particular, bioprocesses that utilize the remarkable properties of enzymes would often benefit from mutants that remain active at temperatures that are either higher or lower than the physiological temperature, while maintaining the biological activity. Many in silico methods have been developed in recent years for predicting the thermodynamic stability of mutant proteins, but very few have focused on thermostability. To bridge this gap, we developed an algorithm for predicting the best descriptor of thermostability, namely the melting temperature Tm, from the protein's sequence and structure. Our method is applicable when the Tm of proteins homologous to the target protein are known. It is based on the design of several temperature-dependent statistical potentials, derived from datasets consisting of either mesostable or thermostable proteins. Linear combinations of these potentials have been shown to yield an estimation of the protein folding free energies at low and high temperatures, and the difference of these energies, a prediction of the melting temperature. This particular construction, that distinguishes between the interactions that contribute more than others to the stability at high temperatures and those that are more stabilizing at low T, gives better performances compared to the standard approach based on T-independent potentials which predict the thermal resistance from the thermodynamic stability. Our method has been tested on 45 proteins of known Tm that belong to 11 homologous families. The standard deviation between experimental and predicted Tm's is equal to 13.6°C in cross validation, and decreases to 8.3°C if the 6 worst predicted proteins are excluded. Possible extensions of our approach are discussed.

  3. Homologous recombination in mammalian cells: effect of p53 and Bcl-2 proteins, replication inhibition and ionizing radiations

    International Nuclear Information System (INIS)

    Saintigny, Yannick

    1999-01-01

    The control of cell cycle, associated with the mechanisms of replication, DNA repair/recombination allows the cells to maintain their genetic integrity. The p53 protein ensures the control of G1/S transition. Its inactivation would allow to initial replication on damaged matrix and lead to the block of replication forks followed by DNA strand breaks, good substrates for recombination. This work shows that the expression of mutant p53 protein stimulates both spontaneous and radio-induced homologous recombination, independently of the control of cell cycle. Moreover, the use of a set of replication inhibitors show that inhibition of the replication elongation stimulates recombination more strongly than the initiation inhibition. Replication arrest by these inhibitors also significantly increases the number of DNA strand breaks. These results highlighted a point of action of p53 protein on the ultimate stages of the homologous recombination mechanism. Lastly, the expression of Bcl-2 protein inhibits apoptosis and increases survival, but specifically inhibits conservative recombination, after radiation as well as in absence of apoptotic stress. The extinction of this mechanism of DNA repair is associated with an increase of mutagenesis. Taken together, these results allow ta consider the maintenance of the genetic stability as a cellular network involving different pathways. A multiple stages model for tumoral progression can be deduced. (author) [fr

  4. Homology modelling and docking analysis of L-lactate dehydrogenase from Streptococcus thermopilus

    Directory of Open Access Journals (Sweden)

    Vukić Vladimir R.

    2016-01-01

    Full Text Available The aim of this research was to create a three-dimensional model of L-lactate dehydrogenase from the main yoghurt starter culture - Streptococcus thermopilus, to analyse its structural features and investigate substrate binding in the active site. NCBI BlastP was used against the Protein Data Bank database in order to identify the template for construction of homology models. Multiple sequence alignment was performed using the program MUSCULE within the UGENE 1.11.3 program. Homology models were constructed using the program Modeller v. 9.17. The obtained 3D model was verified by Ramachandran plots. Molecular docking simulations were performed using the program Surflex-Dock. The highest sequence similarity was observed with L-lactate dehydrogenase from Lactobacillus casei subsp. casei, with 69% identity. Therefore, its structure (PDB ID: 2ZQY:A was selected as a modelling template for homology modelling. Active residues are by sequence similarity predicted: S. thermophilus - HIS181 and S. aureus - HIS179. Binding energy of pyruvate to L-lactate dehydrogenase of S. thermopilus was - 7.874 kcal/mol. Pyruvate in L-lactate dehydrogenase of S. thermopilus makes H bonds with catalytic HIS181 (1.9 Å, as well as with THR235 (3.6 Å. Although our results indicate similar position of substrates between L-lactate dehydrogenase of S. thermopilus and S. aureus, differences in substrate distances and binding energy values could influence the reaction rate. Based on these results, the L-lactate dehydrogenase model proposed here could be used as a guide for further research, such as transition states of the reaction through molecular dynamics. [Projekat Ministarstva nauke Republike Srbije, br. III 46009

  5. Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium salinarum in Survival of DNA Damage.

    Science.gov (United States)

    Evans, Jessica J; Gygli, Patrick E; McCaskill, Julienne; DeVeaux, Linda C

    2018-04-20

    The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic and are encoded on the minichromosomes, and one is uniquely euryarchaeal. Radiation-resistant mutants previously isolated show upregulation of one of the eukaryotic-type RPA genes. Here, we have created deletions in the five RPA operons. These deletion mutants were exposed to DNA-damaging conditions: ionizing radiation, UV radiation, and mitomycin C. Deletion of the euryarchaeal homolog, although not lethal as in Haloferax volcanii , causes severe sensitivity to all of these agents. Deletion of the other RPA/SSB homologs imparts a variable sensitivity to these DNA-damaging agents, suggesting that the different RPA homologs have specialized roles depending on the type of genomic insult encountered.

  6. A macrophage inflammatory protein homolog encoded by guinea pig cytomegalovirus signals via CC chemokine receptor 1

    International Nuclear Information System (INIS)

    Penfold, Mark; Miao Zhenhua; Wang Yu; Haggerty, Shannon; Schleiss, Mark R.

    2003-01-01

    Cytomegaloviruses encode homologs of cellular immune effector proteins, including chemokines (CKs) and CK receptor-like G protein-coupled receptors (GPCRs). Sequence of the guinea pig cytomegalovirus (GPCMV) genome identified an open reading frame (ORF) which predicted a 101 amino acid (aa) protein with homology to the macrophage inflammatory protein (MIP) subfamily of CC (β) CKs, designated GPCMV-MIP. To assess functionality of this CK, recombinant GPCMV-MIP was expressed in HEK293 cells and assayed for its ability to bind to and functionally interact with a variety of GPCRs. Specific signaling was observed with the hCCR1 receptor, which could be blocked with hMIP -1α in competition experiments. Migration assays revealed that GPCMV-MIP was able to induce chemotaxis in hCCR1-L1.2 cells. Antisera raised against a GST-MIP fusion protein immunoprecipitated species of ∼12 and 10 kDa from GPCMV-inoculated tissue culture lysates, and convalescent antiserum from GPCMV-infected animals was immunoreactive with GST-MIP by ELISA assay. These results represent the first substantive in vitro characterization of a functional CC CK encoded by a cytomegalovirus

  7. Protein backbone angle restraints from searching a database for chemical shift and sequence homology

    Energy Technology Data Exchange (ETDEWEB)

    Cornilescu, Gabriel; Delaglio, Frank; Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    1999-03-15

    Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13C{alpha}, 13C{beta}, 13C', 1H{alpha} and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar {phi} and {psi} backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15 deg. Approximately 3% of the predictions made by TALOS are found to be in error.

  8. The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase.

    OpenAIRE

    Caldas , Thérèse; Malki , Abderrahim; Kern , Renée; Abdallah , Jad; Richarme , Gilbert

    2006-01-01

    Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN func...

  9. Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

    Directory of Open Access Journals (Sweden)

    Yushen Du

    2016-11-01

    Full Text Available Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp, we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.

  10. VITAL NMR: using chemical shift derived secondary structure information for a limited set of amino acids to assess homology model accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Brothers, Michael C.; Nesbitt, Anna E.; Hallock, Michael J. [University of Illinois at Urbana-Champaign, Department of Chemistry (United States); Rupasinghe, Sanjeewa G. [University of Illinois at Urbana-Champaign, Department of Cell and Developmental Biology (United States); Tang Ming [University of Illinois at Urbana-Champaign, Department of Chemistry (United States); Harris, Jason; Baudry, Jerome [University of Tennessee, Department of Biochemistry, Cellular and Molecular Biology (United States); Schuler, Mary A. [University of Illinois at Urbana-Champaign, Department of Cell and Developmental Biology (United States); Rienstra, Chad M., E-mail: rienstra@illinois.edu [University of Illinois at Urbana-Champaign, Department of Chemistry (United States)

    2012-01-15

    Homology modeling is a powerful tool for predicting protein structures, whose success depends on obtaining a reasonable alignment between a given structural template and the protein sequence being analyzed. In order to leverage greater predictive power for proteins with few structural templates, we have developed a method to rank homology models based upon their compliance to secondary structure derived from experimental solid-state NMR (SSNMR) data. Such data is obtainable in a rapid manner by simple SSNMR experiments (e.g., {sup 13}C-{sup 13}C 2D correlation spectra). To test our homology model scoring procedure for various amino acid labeling schemes, we generated a library of 7,474 homology models for 22 protein targets culled from the TALOS+/SPARTA+ training set of protein structures. Using subsets of amino acids that are plausibly assigned by SSNMR, we discovered that pairs of the residues Val, Ile, Thr, Ala and Leu (VITAL) emulate an ideal dataset where all residues are site specifically assigned. Scoring the models with a predicted VITAL site-specific dataset and calculating secondary structure with the Chemical Shift Index resulted in a Pearson correlation coefficient (-0.75) commensurate to the control (-0.77), where secondary structure was scored site specifically for all amino acids (ALL 20) using STRIDE. This method promises to accelerate structure procurement by SSNMR for proteins with unknown folds through guiding the selection of remotely homologous protein templates and assessing model quality.

  11. VITAL NMR: Using Chemical Shift Derived Secondary Structure Information for a Limited Set of Amino Acids to Assess Homology Model Accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Brothers, Michael C [University of Illinois, Urbana-Champaign; Nesbitt, Anna E [University of Illinois, Urbana-Champaign; Hallock, Michael J [University of Illinois, Urbana-Champaign; Rupasinghe, Sanjeewa [University of Illinois, Urbana-Champaign; Tang, Ming [University of Illinois, Urbana-Champaign; Harris, Jason B [ORNL; Baudry, Jerome Y [ORNL; Schuler, Mary A [University of Illinois, Urbana-Champaign; Rienstra, Chad M [University of Illinois, Urbana-Champaign

    2011-01-01

    Homology modeling is a powerful tool for predicting protein structures, whose success depends on obtaining a reasonable alignment between a given structural template and the protein sequence being analyzed. In order to leverage greater predictive power for proteins with few structural templates, we have developed a method to rank homology models based upon their compliance to secondary structure derived from experimental solid-state NMR (SSNMR) data. Such data is obtainable in a rapid manner by simple SSNMR experiments (e.g., (13)C-(13)C 2D correlation spectra). To test our homology model scoring procedure for various amino acid labeling schemes, we generated a library of 7,474 homology models for 22 protein targets culled from the TALOS+/SPARTA+ training set of protein structures. Using subsets of amino acids that are plausibly assigned by SSNMR, we discovered that pairs of the residues Val, Ile, Thr, Ala and Leu (VITAL) emulate an ideal dataset where all residues are site specifically assigned. Scoring the models with a predicted VITAL site-specific dataset and calculating secondary structure with the Chemical Shift Index resulted in a Pearson correlation coefficient (-0.75) commensurate to the control (-0.77), where secondary structure was scored site specifically for all amino acids (ALL 20) using STRIDE. This method promises to accelerate structure procurement by SSNMR for proteins with unknown folds through guiding the selection of remotely homologous protein templates and assessing model quality.

  12. DockoMatic 2.0: high throughput inverse virtual screening and homology modeling.

    Science.gov (United States)

    Bullock, Casey; Cornia, Nic; Jacob, Reed; Remm, Andrew; Peavey, Thomas; Weekes, Ken; Mallory, Chris; Oxford, Julia T; McDougal, Owen M; Andersen, Timothy L

    2013-08-26

    DockoMatic is a free and open source application that unifies a suite of software programs within a user-friendly graphical user interface (GUI) to facilitate molecular docking experiments. Here we describe the release of DockoMatic 2.0; significant software advances include the ability to (1) conduct high throughput inverse virtual screening (IVS); (2) construct 3D homology models; and (3) customize the user interface. Users can now efficiently setup, start, and manage IVS experiments through the DockoMatic GUI by specifying receptor(s), ligand(s), grid parameter file(s), and docking engine (either AutoDock or AutoDock Vina). DockoMatic automatically generates the needed experiment input files and output directories and allows the user to manage and monitor job progress. Upon job completion, a summary of results is generated by Dockomatic to facilitate interpretation by the user. DockoMatic functionality has also been expanded to facilitate the construction of 3D protein homology models using the Timely Integrated Modeler (TIM) wizard. The wizard TIM provides an interface that accesses the basic local alignment search tool (BLAST) and MODELER programs and guides the user through the necessary steps to easily and efficiently create 3D homology models for biomacromolecular structures. The DockoMatic GUI can be customized by the user, and the software design makes it relatively easy to integrate additional docking engines, scoring functions, or third party programs. DockoMatic is a free comprehensive molecular docking software program for all levels of scientists in both research and education.

  13. Multi-template homology based structure prediction and molecular docking studies of protein ‘L’ of Zaire ebolavirus (EBOV

    Directory of Open Access Journals (Sweden)

    Jayasree Ganugapati

    2017-01-01

    Full Text Available Ebola is one of the most dangerous pathogenic RNA virus that causes severe hemorrhagic fever in humans and is considered to be a threat to humanity. The RNA genome of EBOV encodes seven proteins viz., glycoprotein (GP, nucleoprotein (NP, RNA-dependent RNA polymerase (protein ‘L’, VP35, VP30, VP40 andVP24. The objective of the present study is to find a suitable inhibitor for protein ‘L’. The large structural protein ‘L’, is made up of 2212 amino acid residues. This protein works as an RNA-dependent RNA polymerase (RdRp and a methyl transferase. It is carried by the virus during the infection as the host mechanisms cannot be used to transcribe the –ss RNA genome of the virus. As the protein is crucial for the replication of the viral genome and no other host enzyme can perform the same function, this viral protein ‘L’ was considered as a potential drug target to design inhibitors. The 3D structure of protein ‘L’ is not available to date. This is a limitation in understanding the protein's function. Hence, the present work is aimed at predicting the first homology-based model of protein ‘L’ and elucidating the function by providing insight into the molecular details of the protein. As there is no drug available for the treatment of EBOV infection our findings play a crucial a role to identify an inhibitor of the protein ‘L’ of EBOV. HTS against ZINC database resulted in identification of few possible inhibitors. Molecular docking studies resulted in finding a suitable inhibitor for protein ‘L’.

  14. α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

    Science.gov (United States)

    Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  15. Bidirectional gene sequences with similar homology to functional proteins of alkane degrading bacterium pseudomonas fredriksbergensis DNA

    International Nuclear Information System (INIS)

    Megeed, A.A.

    2011-01-01

    The potential for two overlapping fragments of DNA from a clone of newly isolated alkanes degrading bacterium Pseudomonas frederiksbergensis encoding sequences with similar homology to two parts of functional proteins is described. One strand contains a sequence with high homology to alkanes monooxygenase (alkB), a member of the alkanes hydroxylase family, and the other strand contains a sequence with some homology to alcohol dehydrogenase gene (alkJ). Overlapping of the genes on opposite strands has been reported in eukaryotic species, and is now reported in a bacterial species. The sequence comparisons and ORFS results revealed that the regulation and the genes organization involved in alkane oxidation represented in Pseudomonas frederiksberghensis varies among the different known alkane degrading bacteria. The alk gene cluster containing homologues to the known alkane monooxygenase (alkB), and rubredoxin (alkG) are oriented in the same direction, whereas alcohol dehydrogenase (alkJ) is oriented in the opposite direction. Such genomes encode messages on both strands of the DNA, or in an overlapping but different reading frames, of the same strand of DNA. The possibility of creating novel genes from pre-existing sequences, known as overprinting, which is a widespread phenomenon in small viruses. Here, the origin and evolution of the gene overlap to bacteriophages belonging to the family Microviridae have been investigated. Such a phenomenon is most widely described in extremely small genomes such as those of viruses or small plasmids, yet here is a unique phenomenon. (author)

  16. MIPS: a database for protein sequences, homology data and yeast genome information.

    Science.gov (United States)

    Mewes, H W; Albermann, K; Heumann, K; Liebl, S; Pfeiffer, F

    1997-01-01

    The MIPS group (Martinsried Institute for Protein Sequences) at the Max-Planck-Institute for Biochemistry, Martinsried near Munich, Germany, collects, processes and distributes protein sequence data within the framework of the tripartite association of the PIR-International Protein Sequence Database (,). MIPS contributes nearly 50% of the data input to the PIR-International Protein Sequence Database. The database is distributed on CD-ROM together with PATCHX, an exhaustive supplement of unique, unverified protein sequences from external sources compiled by MIPS. Through its WWW server (http://www.mips.biochem.mpg.de/ ) MIPS permits internet access to sequence databases, homology data and to yeast genome information. (i) Sequence similarity results from the FASTA program () are stored in the FASTA database for all proteins from PIR-International and PATCHX. The database is dynamically maintained and permits instant access to FASTA results. (ii) Starting with FASTA database queries, proteins have been classified into families and superfamilies (PROT-FAM). (iii) The HPT (hashed position tree) data structure () developed at MIPS is a new approach for rapid sequence and pattern searching. (iv) MIPS provides access to the sequence and annotation of the complete yeast genome (), the functional classification of yeast genes (FunCat) and its graphical display, the 'Genome Browser' (). A CD-ROM based on the JAVA programming language providing dynamic interactive access to the yeast genome and the related protein sequences has been compiled and is available on request. PMID:9016498

  17. Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi

    Directory of Open Access Journals (Sweden)

    D.A. Meireles

    2017-08-01

    Full Text Available Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species. Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr, the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols, but not by β-mercaptoethanol or GSH (monothiols, even in large excess; (ii MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH over hydrogen peroxide; (iii MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13×108 M−1 s−1. Both Cys87 and Cys154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKa value of the Cysp residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Keywords: Ohr/OsmC, Thiol-dependent peroxidases, Phylogeny

  18. Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi.

    Science.gov (United States)

    Meireles, D A; Domingos, R M; Gaiarsa, J W; Ragnoni, E G; Bannitz-Fernandes, R; da Silva Neto, J F; de Souza, R F; Netto, L E S

    2017-08-01

    Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by β-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×10 8 M -1 s -1 ). Both Cys 87 and Cys 154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pK a value of the Cys p residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Copyright © 2017. Published by Elsevier B.V.

  19. RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair

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    Mohammad A.M. Ali

    2018-01-01

    Full Text Available Summary: Ring1-YY1-binding protein (RYBP is a member of the non-canonical polycomb repressive complex 1 (PRC1, and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs, we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding. : Ali et al. find that RYBP binds K63-linked ubiquitin chains and is removed from DNA damage sites. This K63-ubiquitin binding allows RYBP to hinder the recruitment of BRCA1 and Rad51 to DNA double-strand breaks, thus inhibiting homologous recombination repair. Accordingly, cancer cells expressing high RYBP are more sensitive to DNA-damaging therapies. Keywords: DNA damage response, homologous recombination, ubiquitylation, RYBP, polycomb proteins, double-strand break repair, chromatin, histone modification

  20. Inhibition of tumor metastasis by a growth factor receptor bound protein 2 Src homology 2 domain-binding antagonist.

    Science.gov (United States)

    Giubellino, Alessio; Gao, Yang; Lee, Sunmin; Lee, Min-Jung; Vasselli, James R; Medepalli, Sampath; Trepel, Jane B; Burke, Terrence R; Bottaro, Donald P

    2007-07-01

    Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

  1. Establishing homology between mitochondrial calcium uniporters, prokaryotic magnesium channels and chlamydial IncA proteins.

    Science.gov (United States)

    Lee, Andre; Vastermark, Ake; Saier, Milton H

    2014-08-01

    Mitochondrial calcium uniporters (MCUs) (TC no. 1.A.77) are oligomeric channel proteins found in the mitochondrial inner membrane. MCUs have two well-conserved transmembrane segments (TMSs), connected by a linker, similar to bacterial MCU homologues. These proteins and chlamydial IncA proteins (of unknown function; TC no. 9.B.159) are homologous to prokaryotic Mg(2+) transporters, AtpI and AtpZ, based on comparison scores of up to 14.5 sds. A phylogenetic tree containing all of these proteins showed that the AtpZ proteins cluster coherently as a subset within the large and diverse AtpI cluster, which branches separately from the MCUs and IncAs, both of which cluster coherently. The MCUs and AtpZs share the same two TMS topology, but the AtpIs have four TMSs, and IncAs can have either two (most frequent) or four (less frequent) TMSs. Binary alignments, comparison scores and motif analyses showed that TMSs 1 and 2 align with TMSs 3 and 4 of the AtpIs, suggesting that the four TMS AtpI proteins arose via an intragenic duplication event. These findings establish an evolutionary link interconnecting eukaryotic and prokaryotic Ca(2+) and Mg(2+) transporters with chlamydial IncAs, and lead us to suggest that all members of the MCU superfamily, including IncAs, function as divalent cation channels. © 2014 The Authors.

  2. Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion

    International Nuclear Information System (INIS)

    Karim, Shahid; Ramakrishnan, Vijay G.; Tucker, James S.; Essenberg, Richard C.; Sauer, John R.

    2004-01-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in 'knocking down' nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands

  3. COGcollator: a web server for analysis of distant relationships between homologous protein families.

    Science.gov (United States)

    Dibrova, Daria V; Konovalov, Kirill A; Perekhvatov, Vadim V; Skulachev, Konstantin V; Mulkidjanian, Armen Y

    2017-11-29

    The Clusters of Orthologous Groups (COGs) of proteins systematize evolutionary related proteins into specific groups with similar functions. However, the available databases do not provide means to assess the extent of similarity between the COGs. We intended to provide a method for identification and visualization of evolutionary relationships between the COGs, as well as a respective web server. Here we introduce the COGcollator, a web tool for identification of evolutionarily related COGs and their further analysis. We demonstrate the utility of this tool by identifying the COGs that contain distant homologs of (i) the catalytic subunit of bacterial rotary membrane ATP synthases and (ii) the DNA/RNA helicases of the superfamily 1. This article was reviewed by Drs. Igor N. Berezovsky, Igor Zhulin and Yuri Wolf.

  4. A theoretical model of the tridimensional structure of Bacillus thuringiensis subsp. medellin Cry 11Bb toxin deduced by homology modelling

    Directory of Open Access Journals (Sweden)

    Gutierrez Pablo

    2001-01-01

    Full Text Available Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp. medellin during its stationary phase; this ¶-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control. Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin. The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins. In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation. This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

  5. Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities.

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    Jaimie-Leigh Jonker

    Full Text Available Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins 'sticky' has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes. It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa. Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7-16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes. Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18-26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa are more conserved within barnacles than others (20 kDa.

  6. HHR23B, a human RAD23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.

    NARCIS (Netherlands)

    K. Sugasawa (Kaoru); C. Masutani (Chikahide); A. Uchida; T. Maekawa; P.J. van der Spek (Peter); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan); F. Hanaoka (Fumio)

    1996-01-01

    textabstractA protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces

  7. Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides.

    Science.gov (United States)

    Moutal, Aubin; Li, Wennan; Wang, Yue; Ju, Weina; Luo, Shizhen; Cai, Song; François-Moutal, Liberty; Perez-Miller, Samantha; Hu, Jackie; Dustrude, Erik T; Vanderah, Todd W; Gokhale, Vijay; Khanna, May; Khanna, Rajesh

    2017-02-05

    N-type voltage-gated calcium (Ca v 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Ca v 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Ca v 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Ca v 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Ca v 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. © 2017 The British Pharmacological Society.

  8. Improving model construction of profile HMMs for remote homology detection through structural alignment

    Directory of Open Access Journals (Sweden)

    Zaverucha Gerson

    2007-11-01

    Full Text Available Abstract Background Remote homology detection is a challenging problem in Bioinformatics. Arguably, profile Hidden Markov Models (pHMMs are one of the most successful approaches in addressing this important problem. pHMM packages present a relatively small computational cost, and perform particularly well at recognizing remote homologies. This raises the question of whether structural alignments could impact the performance of pHMMs trained from proteins in the Twilight Zone, as structural alignments are often more accurate than sequence alignments at identifying motifs and functional residues. Next, we assess the impact of using structural alignments in pHMM performance. Results We used the SCOP database to perform our experiments. Structural alignments were obtained using the 3DCOFFEE and MAMMOTH-mult tools; sequence alignments were obtained using CLUSTALW, TCOFFEE, MAFFT and PROBCONS. We performed leave-one-family-out cross-validation over super-families. Performance was evaluated through ROC curves and paired two tailed t-test. Conclusion We observed that pHMMs derived from structural alignments performed significantly better than pHMMs derived from sequence alignment in low-identity regions, mainly below 20%. We believe this is because structural alignment tools are better at focusing on the important patterns that are more often conserved through evolution, resulting in higher quality pHMMs. On the other hand, sensitivity of these tools is still quite low for these low-identity regions. Our results suggest a number of possible directions for improvements in this area.

  9. Improving model construction of profile HMMs for remote homology detection through structural alignment.

    Science.gov (United States)

    Bernardes, Juliana S; Dávila, Alberto M R; Costa, Vítor S; Zaverucha, Gerson

    2007-11-09

    Remote homology detection is a challenging problem in Bioinformatics. Arguably, profile Hidden Markov Models (pHMMs) are one of the most successful approaches in addressing this important problem. pHMM packages present a relatively small computational cost, and perform particularly well at recognizing remote homologies. This raises the question of whether structural alignments could impact the performance of pHMMs trained from proteins in the Twilight Zone, as structural alignments are often more accurate than sequence alignments at identifying motifs and functional residues. Next, we assess the impact of using structural alignments in pHMM performance. We used the SCOP database to perform our experiments. Structural alignments were obtained using the 3DCOFFEE and MAMMOTH-mult tools; sequence alignments were obtained using CLUSTALW, TCOFFEE, MAFFT and PROBCONS. We performed leave-one-family-out cross-validation over super-families. Performance was evaluated through ROC curves and paired two tailed t-test. We observed that pHMMs derived from structural alignments performed significantly better than pHMMs derived from sequence alignment in low-identity regions, mainly below 20%. We believe this is because structural alignment tools are better at focusing on the important patterns that are more often conserved through evolution, resulting in higher quality pHMMs. On the other hand, sensitivity of these tools is still quite low for these low-identity regions. Our results suggest a number of possible directions for improvements in this area.

  10. Neural network and SVM classifiers accurately predict lipid binding proteins, irrespective of sequence homology.

    Science.gov (United States)

    Bakhtiarizadeh, Mohammad Reza; Moradi-Shahrbabak, Mohammad; Ebrahimi, Mansour; Ebrahimie, Esmaeil

    2014-09-07

    Due to the central roles of lipid binding proteins (LBPs) in many biological processes, sequence based identification of LBPs is of great interest. The major challenge is that LBPs are diverse in sequence, structure, and function which results in low accuracy of sequence homology based methods. Therefore, there is a need for developing alternative functional prediction methods irrespective of sequence similarity. To identify LBPs from non-LBPs, the performances of support vector machine (SVM) and neural network were compared in this study. Comprehensive protein features and various techniques were employed to create datasets. Five-fold cross-validation (CV) and independent evaluation (IE) tests were used to assess the validity of the two methods. The results indicated that SVM outperforms neural network. SVM achieved 89.28% (CV) and 89.55% (IE) overall accuracy in identification of LBPs from non-LBPs and 92.06% (CV) and 92.90% (IE) (in average) for classification of different LBPs classes. Increasing the number and the range of extracted protein features as well as optimization of the SVM parameters significantly increased the efficiency of LBPs class prediction in comparison to the only previous report in this field. Altogether, the results showed that the SVM algorithm can be run on broad, computationally calculated protein features and offers a promising tool in detection of LBPs classes. The proposed approach has the potential to integrate and improve the common sequence alignment based methods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Molecular mechanism of protein assembly on DNA double-strand breaks in the non-homologous end-joining pathway

    International Nuclear Information System (INIS)

    Yano, Ken-ichi; Morotomi-Yano, Keiko; Adachi, Noritaka; Akiyama, Hidenori

    2009-01-01

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Upon DSB induction, a living cell quickly activates the NHEJ pathway comprising of multiple molecular events. However, it has been difficult to analyze the initial phase of DSB responses in living cells, primarily due to technical limitations. Recent advances in real-time imaging and site-directed DSB induction using laser microbeam allow us to monitor the spatiotemporal dynamics of NHEJ factors in the immediate-early phase after DSB induction. These new approaches, together with the use of cell lines deficient in each essential NHEJ factor, provide novel mechanistic insights into DSB recognition and protein assembly on DSBs in the NHEJ pathway. In this review, we provide an overview of recent progresses in the imaging analyses of the NHEJ core factors. These studies strongly suggest that the NHEJ core factors are pre-assembled into a large complex on DSBs prior to the progression of the biochemical reactions in the NHEJ pathway. Instead of the traditional step-by-step assembly model from the static view of NHEJ, a novel model for dynamic protein assembly in the NHEJ pathway is proposed. This new model provides important mechanistic insights into the protein assembly at DSBs and the regulation of DSB repair. (author)

  12. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of

  13. Autonomously folding protein fragments reveal differences in the energy landscapes of homologous RNases H.

    Directory of Open Access Journals (Sweden)

    Laura E Rosen

    Full Text Available An important approach to understanding how a protein sequence encodes its energy landscape is to compare proteins with different sequences that fold to the same general native structure. In this work, we compare E. coli and T. thermophilus homologs of the protein RNase H. Using protein fragments, we create equilibrium mimics of two different potential partially-folded intermediates (I(core and I(core+1 hypothesized to be present on the energy landscapes of these two proteins. We observe that both T. thermophilus RNase H (ttRNH fragments are folded and have distinct stabilities, indicating that both regions are capable of autonomous folding and that both intermediates are present as local minima on the ttRNH energy landscape. In contrast, the two E. coli RNase H (ecRNH fragments have very similar stabilities, suggesting that the presence of additional residues in the I(core+1 fragment does not affect the folding or structure as compared to I(core. NMR experiments provide additional evidence that only the I(core intermediate is populated by ecRNH. This is one of the biggest differences that has been observed between the energy landscapes of these two proteins. Additionally, we used a FRET experiment in the background of full-length ttRNH to specifically monitor the formation of the I(core+1 intermediate. We determine that the ttRNH I(core+1 intermediate is likely the intermediate populated prior to the rate-limiting barrier to global folding, in contrast to E. coli RNase H for which I(core is the folding intermediate. This result provides new insight into the nature of the rate-limiting barrier for the folding of RNase H.

  14. HOMOLOGY BETWEEN SEGMENTS OF HUMAN HEMOSTATIC PROTEINS AND PROTEINS OF VIRUSES WHICH CAUSE ACUTE RESPIRATORY INFECTIONS OR DISEASES WITH SIMILAR SYMPTOMS

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    I. N. Zhilinskaya

    2017-01-01

    Full Text Available Objectives: To identify homologous segments of human hemostatic and viral proteins and to assess the role of human hemostatic proteins in viral replication. Materials and Methods: The following viruses were chosen for comparison: influenza B (B/Astrakhan/2/2017, coronaviruses (Hcov229E and SARS-Co, type 1 adenovirus (adenoid 71, measles (ICHINOSE-BA and rubella (Therien. The primary structures of viral proteins and 41 human hemostatic proteins were obtained from open–access www.ncbi.nlm.nih. gov and www.nextprot.org databases, respectively. Sequence homology was determined by comparing 12-amino-acid segments. Those sequences identical in ≥ 8 positions were considered homologous. Results: The analysis shows that viral proteins contain segments which mimic a number of human hemostatic proteins. Most of these segments, except those of adenovirus proteins, are homologous with coagulation factors. The increase in viral virulence, as in case of SARS-Co, correlates with an increased number of segments homologous with hemostatic proteins. Conclusion: Hemostasis plays an important role in viral replication and pathogenesis. The conclusion is consistent with the literature data about the relationship of hemostasis and inflammatory response to viral infections.

  15. Genomic clustering and homology between HET-S and the NWD2 STAND protein in various fungal genomes.

    Directory of Open Access Journals (Sweden)

    Asen Daskalov

    Full Text Available BACKGROUND: Prions are infectious proteins propagating as self-perpetuating amyloid polymers. The [Het-s] prion of Podospora anserina is involved in a cell death process associated with non-self recognition. The prion forming domain (PFD of HET-s adopts a β-solenoid amyloid structure characterized by the two fold repetition of an elementary triangular motif. [Het-s] induces cell death when interacting with HET-S, an allelic variant of HET-s. When templated by [Het-s], HET-S undergoes a trans-conformation, relocates to the cell membrane and induces toxicity. METHODOLOGY/PRINCIPAL FINDINGS: Here, comparing HET-s homologs from different species, we devise a consensus for the HET-s elementary triangular motif. We use this motif to screen genomic databases and find a match to the N-terminus of NWD2, a STAND protein, encoded by the gene immediately adjacent to het-S. STAND proteins are signal transducing ATPases which undergo ligand-induced oligomerisation. Homology modelling predicts that the NWD2 N-terminal region adopts a HET-s-like fold. We propose that upon NWD2 oligomerisation, these N-terminal extensions adopt the β-solenoid fold and template HET-S to adopt the amyloid fold and trigger toxicity. We extend this model to a putative prion, the σ infectious element in Nectria haematococca, because the s locus controlling propagation of σ also encodes a STAND protein and displays analogous features. Comparative genomic analyses indicate evolutionary conservation of these STAND/prion-like gene pairs, identify a number of novel prion candidates and define, in addition to the HET-s PFD motif, two distinct, novel putative PFD-like motifs. CONCLUSIONS/SIGNIFICANCE: We suggest the existence, in the fungal kingdom, of a widespread and evolutionarily conserved mode of signal transduction based on the transmission of an amyloid-fold from a NOD-like STAND receptor protein to an effector protein.

  16. The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase.

    Science.gov (United States)

    Caldas, Thérèse; Malki, Abderrahim; Kern, Renée; Abdallah, Jad; Richarme, Gilbert

    2006-05-12

    Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.

  17. Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST

    Directory of Open Access Journals (Sweden)

    Nalin CW Goonesekere

    2009-06-01

    Full Text Available Nalin CW GoonesekereDepartment of Chemistry and Biochemistry, University of Northern iowa, Cedar Falls, IA, USAAbstract: The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP database. We show that when incorporated into the homology search algorithms BLAST and PSI-blaST, the structure-based substitution matrices enhance the efficacy of detecting remote homologs. Keywords: computational biology, protein homology, amino acid substitution matrix, protein structure

  18. In silico analysis of candidate proteins sharing homology with Streptococcus agalactiae proteins and their role in male infertility.

    Science.gov (United States)

    Parida, Rajeshwari; Samanta, Luna

    2017-02-01

    Leukocytospermia is a physiologic condition defined as human semen with a leukocyte count of >1 x 10 6 cells/ml that is often correlated with male infertility. Moreover, bacteriospermia has been associated with leukocytospermia ultimately leading to male infertility. We have found that semen samples with >1 x 10 6 /ml leukocytes and/or bacteriospermia have oxidative predominance as evidenced by augmented protein carbonyl and lipid peroxidation status of the semen which is implicated in sperm dysfunction. It has been reported that Streptococcus agalactiae is present in bacteriospermic samples. Previous research has shown that human leukocyte antigen beta chain paralog (HLA-DRB) alleles interact best with the infected sperm cells rather than the non-infected cells. Little is known about the interaction of major histocompatibility complex (MHC) present on leukocytes with the sperm upon bacterial infection and how it induces an immunological response which we have addressed by epitope mapping. Therefore, we examined MHC class II derived bacterial peptides which might have human sperm-related functional aspects. Twenty-two S. agalactiae proteins were obtained from PUBMED protein database for our study. Protein sequences with more than two accession numbers were aligned using CLUSTAL Omega to check their conservation pattern. Each protein sequence was then analyzed for T-cell epitope prediction against HLA-DRB alleles using the immune epitope database (IEDB) analysis tool. Out of a plethora of peptides obtained from this analysis, peptides corresponding to proteins of interest such as DNA binding response regulator, hyaluronate lyase and laminin binding protein were screened against the human proteome using Blastp. Interestingly, we have found bacterial peptides sharing homology with human peptides deciphering some of the important sperm functions. Antibodies raised against these probable bacterial antigens of fertility will not only help us understand the mechanism of

  19. Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen.

    Science.gov (United States)

    Collier, Thomas A; Nash, Anthony; Birch, Helen L; de Leeuw, Nora H

    2018-02-15

    Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule's size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules.

  20. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    International Nuclear Information System (INIS)

    Chang, Soo-Ik; Hammes, G.G.

    1989-01-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the β-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution

  1. Protein homology network families reveal step-wise diversification of Type III and Type IV secretion systems.

    Directory of Open Access Journals (Sweden)

    Duccio Medini

    2006-12-01

    Full Text Available From the analysis of 251 prokaryotic genomes stored in public databases, the 761,260 deduced proteins were used to reconstruct a complete set of bacterial proteic families. Using the new Overlap algorithm, we have partitioned the Protein Homology Network (PHN, where the proteins are the nodes and the links represent homology relationships. The algorithm identifies the densely connected regions of the PHN that define the families of homologous proteins, here called PHN-Families, recognizing the phylogenetic relationships embedded in the network. By direct comparison with a manually curated dataset, we assessed that this classification algorithm generates data of quality similar to a human expert. Then, we explored the network to identify families involved in the assembly of Type III and Type IV secretion systems (T3SS and T4SS. We noticed that, beside a core of conserved functions (eight proteins for T3SS, seven for T4SS, a variable set of accessory components is always present (one to nine for T3SS, one to five for T4SS. Each member of the core corresponds to a single PHN-Family, while accessory proteins are distributed among different pure families. The PHN-Family classification suggests that T3SS and T4SS have been assembled through a step-wise, discontinuous process, by complementing the conserved core with subgroups of nonconserved proteins. Such genetic modules, independently recruited and probably tuned on specific effectors, contribute to the functional specialization of these organelles to different microenvironments.

  2. The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins.

    Science.gov (United States)

    Lin, A; McNally, J; Wool, I G

    1983-09-10

    The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.

  3. Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily

    Science.gov (United States)

    Lukk, Tiit; Sakai, Ayano; Kalyanaraman, Chakrapani; Brown, Shoshana D.; Imker, Heidi J.; Song, Ling; Fedorov, Alexander A.; Fedorov, Elena V.; Toro, Rafael; Hillerich, Brandan; Seidel, Ronald; Patskovsky, Yury; Vetting, Matthew W.; Nair, Satish K.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, John A.; Jacobson, Matthew P.

    2012-01-01

    The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion. PMID:22392983

  4. Conserved Functional Motifs and Homology Modeling to Predict Hidden Moonlighting Functional Sites

    KAUST Repository

    Wong, Aloysius Tze; Gehring, Christoph A; Irving, Helen R.

    2015-01-01

    Moonlighting functional centers within proteins can provide them with hitherto unrecognized functions. Here, we review how hidden moonlighting functional centers, which we define as binding sites that have catalytic activity or regulate protein function in a novel manner, can be identified using targeted bioinformatic searches. Functional motifs used in such searches include amino acid residues that are conserved across species and many of which have been assigned functional roles based on experimental evidence. Molecules that were identified in this manner seeking cyclic mononucleotide cyclases in plants are used as examples. The strength of this computational approach is enhanced when good homology models can be developed to test the functionality of the predicted centers in silico, which, in turn, increases confidence in the ability of the identified candidates to perform the predicted functions. Computational characterization of moonlighting functional centers is not diagnostic for catalysis but serves as a rapid screening method, and highlights testable targets from a potentially large pool of candidates for subsequent in vitro and in vivo experiments required to confirm the functionality of the predicted moonlighting centers.

  5. Conserved Functional Motifs and Homology Modeling to Predict Hidden Moonlighting Functional Sites

    KAUST Repository

    Wong, Aloysius Tze

    2015-06-09

    Moonlighting functional centers within proteins can provide them with hitherto unrecognized functions. Here, we review how hidden moonlighting functional centers, which we define as binding sites that have catalytic activity or regulate protein function in a novel manner, can be identified using targeted bioinformatic searches. Functional motifs used in such searches include amino acid residues that are conserved across species and many of which have been assigned functional roles based on experimental evidence. Molecules that were identified in this manner seeking cyclic mononucleotide cyclases in plants are used as examples. The strength of this computational approach is enhanced when good homology models can be developed to test the functionality of the predicted centers in silico, which, in turn, increases confidence in the ability of the identified candidates to perform the predicted functions. Computational characterization of moonlighting functional centers is not diagnostic for catalysis but serves as a rapid screening method, and highlights testable targets from a potentially large pool of candidates for subsequent in vitro and in vivo experiments required to confirm the functionality of the predicted moonlighting centers.

  6. Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

    Science.gov (United States)

    Vergara-Jaque, Ariela; Fenollar-Ferrer, Cristina; Kaufmann, Desirée; Forrest, Lucy R

    2015-01-01

    Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also

  7. Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms

    Directory of Open Access Journals (Sweden)

    Cristina eFenollar Ferrer

    2015-09-01

    Full Text Available Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to either the outside or inside of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (asymmetry of these systems has been successfully used as a bioinformatic tool, called repeat-swap modeling to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that

  8. Cluster based on sequence comparison of homologous proteins of 95 organism species - Gclust Server | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Gclust Server Cluster based on sequence comparison of homologous proteins of 95 organism spe...cies Data detail Data name Cluster based on sequence comparison of homologous proteins of 95 organism specie...istory of This Database Site Policy | Contact Us Cluster based on sequence compariso

  9. Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining

    Directory of Open Access Journals (Sweden)

    Rebecca Cook

    2015-03-01

    Full Text Available Deficiencies in DNA double-strand break (DSB repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1 is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ. Support of cNHEJ involves a mechanism independent of RB1’s cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution.

  10. Silencing the lettuce homologs of small rubber particle protein does not influence natural rubber biosynthesis in lettuce (Lactuca sativa).

    Science.gov (United States)

    Chakrabarty, Romit; Qu, Yang; Ro, Dae-Kyun

    2015-05-01

    Natural rubber, cis-1,4-polyisoprene, is an important raw material in chemical industries, but its biosynthetic mechanism remains elusive. Natural rubber is known to be synthesized in rubber particles suspended in laticifer cells in the Brazilian rubber tree (Hevea brasiliensis). In the rubber tree, rubber elongation factor (REF) and its homolog, small rubber particle protein (SRPP), were found to be the most abundant proteins in rubber particles, and they have been implicated in natural rubber biosynthesis. As lettuce (Lactuca sativa) can synthesize natural rubber, we utilized this annual, transformable plant to examine in planta roles of the lettuce REF/SRPP homologs by RNA interference. Among eight lettuce REF/SRPP homologs identified, transcripts of two genes (LsSRPP4 and LsSRPP8) accounted for more than 90% of total transcripts of REF/SRPP homologs in lettuce latex. LsSRPP4 displays a typical primary protein sequence as other REF/SRPP, while LsSRPP8 is twice as long as LsSRPP4. These two major LsSRPP transcripts were individually and simultaneously silenced by RNA interference, and relative abundance, polymer molecular weight, and polydispersity of natural rubber were analyzed from the LsSRPP4- and LsSRPP8-silenced transgenic lettuce. Despite previous data suggesting the implications of REF/SRPP in natural rubber biosynthesis, qualitative and quantitative alterations of natural rubber could not be observed in transgenic lettuce lines. It is concluded that lettuce REF/SRPP homologs are not critically important proteins in natural rubber biosynthesis in lettuce. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Homology modeling of the serotonin transporter: Insights into the primary escitalopram-binding Site

    DEFF Research Database (Denmark)

    Jørgensen, Anne Marie; Tagmose, L.; Jørgensen, A.M.M.

    2007-01-01

    -ray structure of the closely related amino acid transporter, Aquifex aeolicus leucine transporter (LeuT), provides an opportunity to develop a three-dimensional model of the structure of SERT. We present herein a homology model of SERT using LeuT as the template and containing escitalopram as a bound ligand...

  12. How to Choose the Suitable Template for Homology Modelling of GPCRs: 5-HT7 Receptor as a Test Case.

    Science.gov (United States)

    Shahaf, Nir; Pappalardo, Matteo; Basile, Livia; Guccione, Salvatore; Rayan, Anwar

    2016-09-01

    G protein-coupled receptors (GPCRs) are a super-family of membrane proteins that attract great pharmaceutical interest due to their involvement in almost every physiological activity, including extracellular stimuli, neurotransmission, and hormone regulation. Currently, structural information on many GPCRs is mainly obtained by the techniques of computer modelling in general and by homology modelling in particular. Based on a quantitative analysis of eighteen antagonist-bound, resolved structures of rhodopsin family "A" receptors - also used as templates to build 153 homology models - it was concluded that a higher sequence identity between two receptors does not guarantee a lower RMSD between their structures, especially when their pair-wise sequence identity (within trans-membrane domain and/or in binding pocket) lies between 25 % and 40 %. This study suggests that we should consider all template receptors having a sequence identity ≤50 % with the query receptor. In fact, most of the GPCRs, compared to the currently available resolved structures of GPCRs, fall within this range and lack a correlation between structure and sequence. When testing suitability for structure-based drug design, it was found that choosing as a template the most similar resolved protein, based on sequence resemblance only, led to unsound results in many cases. Molecular docking analyses were carried out, and enrichment factors as well as attrition rates were utilized as criteria for assessing suitability for structure-based drug design. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

    OpenAIRE

    Kadamur, Ganesh; Ross, Elliott M.

    2016-01-01

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in...

  14. Cellulase enzyme: Homology modeling, binding site identification and molecular docking

    Science.gov (United States)

    Selvam, K.; Senbagam, D.; Selvankumar, T.; Sudhakar, C.; Kamala-Kannan, S.; Senthilkumar, B.; Govarthanan, M.

    2017-12-01

    Cellulase is an enzyme that degrades the linear polysaccharide like cellulose into glucose by breaking the β-1,4- glycosidic bonds. These enzymes are the third largest enzymes with a great potential towards the ethanol production and play a vital role in degrading the biomass. The production of ethanol depends upon the ability of the cellulose to utilize the wide range of substrates. In this study, the 3D structure of cellulase from Acinetobacter sp. was modeled by using Modeler 9v9 and validated by Ramachandran plot. The accuracy of the predicted 3D structure was checked using Ramachandran plot analysis showed that 81.1% in the favored region, compatibility of an atomic model (3D) with amino acid sequence (1D) for the model was observed as 78.21% and 49.395% for Verify 3D and ERRAT at SAVES server. As the binding efficacy with the substrate might suggests the choice of the substrate as carbon and nitrogen sources, the cellobiose, cellotetraose, cellotetriose and laminaribiose were employed in the docking studies. The docking of cellobiose, cellotetraose, cellotetriose and laminaribiose with cellulase exhibited the binding energy of -6.1523 kJ/mol, -7.8759 kJ/mol,-6.1590 kJ/mol and -6.7185 kJ/mol, respectively. These docking studies revealed that cellulase has the greater potential towards the cellotetraose as a substrate for the high yield of ethanol.

  15. Molecular modeling used to evaluate CYP2C9-dependent metabolism: homology modeling, molecular dynamics and docking simulations.

    Science.gov (United States)

    Mendieta-Wejebe, Jessica E; Correa-Basurto, José; García-Segovia, Erika M; Ceballos-Cancino, Gisela; Rosales-Hernández, Martha C

    2011-07-01

    Cytochrome P450 (CYP) 2C9 is the principal isoform of the CYP2C subfamily in the human liver and is involved in the oxidation of several endogenous and xenobiotic compounds, including many therapeutic drugs. The metabolism of drugs by CYP2C9 can yield either safe or toxic products, which may be related to the recognition and binding modes of the substrates to this isoform. These interactions can be studied using in silico methods such as quantum chemistry, molecular dynamics and docking simulations, which can also be useful for predicting the structure of metabolites. In these types of studies, the ligand and the protein must be tridimensional models; thus, the protein can be built by homology modeling or retrieved from the Protein Data Bank. Therefore, the current review emphasizes the importance of using in silico methods to predict the metabolism of CYP2C9 because these computational tools have allowed the description of the principal characteristics of the active site of this isoform at the molecular level and the chemical properties of its ligands.

  16. Amino acid sequences mediating vascular cell adhesion molecule 1 binding to integrin alpha 4: homologous DSP sequence found for JC polyoma VP1 coat protein

    Directory of Open Access Journals (Sweden)

    Michael Andrew Meyer

    2013-07-01

    Full Text Available The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4 to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3. For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  17. [Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

    Science.gov (United States)

    Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan

    2017-03-01

    Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A 2 +(A 1 -A 2 )/(1+(x/x 0 ) p ) (A 1 =1.28; A 2 =-0.066; x 0 =12560.75; p=0.74) with a correlation coefficient (R 2 ) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

  18. Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

    International Nuclear Information System (INIS)

    Mao Li; Bryantsev, Anton L.; Chechenova, Maria B.; Shelden, Eric A.

    2005-01-01

    Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features of mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function

  19. Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST.

    Science.gov (United States)

    Goonesekere, Nalin Cw

    2009-01-01

    The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP) database. We show that when incorporated into the homology search algorithms BLAST and PSI-blast, the structure-based substitution matrices enhance the efficacy of detecting remote homologs.

  20. Homologous high-throughput expression and purification of highly conserved E coli proteins

    Directory of Open Access Journals (Sweden)

    Duchmann Rainer

    2007-06-01

    Full Text Available Abstract Background Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD. Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. Results As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP, N-utilizing substance A (NusA, or glutathione S-transferase (GST to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS. Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500

  1. Modeling complexes of modeled proteins.

    Science.gov (United States)

    Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A

    2017-03-01

    Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C α RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold*

    Science.gov (United States)

    Tonkin, Michelle L.; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

    2014-01-01

    Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation. PMID:24675080

  3. Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

    Science.gov (United States)

    Ha, Sookhee N; Hey, Pat J; Ransom, Rick W; Harrell, C Meacham; Murphy, Kathryn L; Chang, Ray; Chen, Tsing-Bau; Su, Dai-Shi; Markowitz, M Kristine; Bock, Mark G; Freidinger, Roger M; Hess, Fred J

    2005-05-27

    We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

  4. Homology modeling and in silico prediction of Ulcerative colitis associated polymorphisms of NOD1.

    Science.gov (United States)

    Majumdar, Ishani; Nagpal, Isha; Paul, Jaishree

    2017-10-01

    Cytosolic pattern recognition receptors play key roles in innate immune response. Nucleotide binding and oligomerisation domain containing protein 1 (NOD1) belonging to the Nod-like receptor C (NLRC) sub-family of Nod-like receptors (NLRs) is important for detection and clearance of intra-cellular Gram negative bacteria. NOD1 is involved in activation of pro-inflammatory pathways. Limited structural data is available for NOD1. Using different templates for each domain of NOD1, we determined the full-length homology model of NOD1. ADP binding amino acids within the nucleotide binding domain (NBD) of NOD1 were also predicted. Key residues in inter-domain interaction were identified by sequence comparison with Oryctolagus cuniculus NOD2, a related protein. Interactions between NBD and winged helix domain (WHD) were found to be conserved in NOD1. Functional and structural effect of single nucleotide polymorphisms within the NOD1 NBD domain associated with susceptibility risk to Ulcerative colitis (UC), an inflammatory disorder of the colon was evaluated by in silico studies. Mutations W219R and L349P were predicted to be damaging and disease associated by prediction programs SIFT, PolyPhen2, PANTHER, SNP&GO, PhD SNP and SNAP2. We further validated the effect of W219R and L349P mutation on NOD1 function in vitro. Elevated mRNA expression of pro-inflammatory cytokines IL8 and IL-1β was seen as compared to the wild type NOD1 in intestinal epithelial cell line HT29 when stimulated with NOD1 ligand. Thus, these mutations may indeed have a bearing on pathogenesis of inflammation during UC. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots.

    Science.gov (United States)

    Stergiopoulos, Ioannis; van den Burg, Harrold A; Okmen, Bilal; Beenen, Henriek G; van Liere, Sabine; Kema, Gert H J; de Wit, Pierre J G M

    2010-04-20

    Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis, the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis, one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

  6. Novel protein interactions with an actin homolog (MreB) of Helicobacter pylori determined by bacterial two-hybrid system.

    Science.gov (United States)

    Zepeda Gurrola, Reyna Cristina; Fu, Yajuan; Rodríguez Luna, Isabel Cristina; Benítez Cardoza, Claudia Guadalupe; López López, María de Jesús; López Vidal, Yolanda; Gutíerrez, Germán Rubén Aguilar; Rodríguez Pérez, Mario A; Guo, Xianwu

    2017-08-01

    The bacterium Helicobacter pylori infects more than 50% of the world population and causes several gastroduodenal diseases, including gastric cancer. Nevertheless, we still need to explore some protein interactions that may be involved in pathogenesis. MreB, an actin homolog, showed some special characteristics in previous studies, indicating that it could have different functions. Protein functions could be realized via protein-protein interactions. In the present study, the MreB protein from H. pylori 26695 fused with two tags 10×His and GST in tandem was overexpressed and purified from Escherchia coli. The purified recombinant protein was used to perform a pull-down assay with H. pylori 26695 cell lysate. The pulled-down proteins were identified by mass spectrometry (MALDI-TOF), in which the known important proteins related to morphogenesis were absent but several proteins related to pathogenesis process were observed. The bacterial two-hybrid system was further used to evaluate the protein interactions and showed that new interactions of MreB respectively with VacA, UreB, HydB, HylB and AddA were confirmed but the interaction MreB-MreC was not validated. These results indicated that the protein MreB in H. pylori has a distinct interactome, does not participate in cell morphogenesis via MreB-MreC but could be related to pathogenesis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

    Science.gov (United States)

    Brown, Simon David; Jarosinska, Olga Dorota; Lorenz, Alexander

    2018-03-17

    Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

  8. Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

    Directory of Open Access Journals (Sweden)

    Ravi Chandra Yandamuri

    2014-10-01

    Full Text Available eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

  9. Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

    Science.gov (United States)

    Jones, Robert T; Bakker, Saskia E; Stone, Deborah; Shuttleworth, Sally N; Boundy, Sam; McCart, Caroline; Daborn, Phillip J; ffrench-Constant, Richard H; van den Elsen, Jean M H

    2010-10-01

    Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.

  10. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    International Nuclear Information System (INIS)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H.

    1988-01-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10 6 independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I

  11. Construction and validation of a homology model of the human voltage-gated proton channel hHV1.

    Science.gov (United States)

    Kulleperuma, Kethika; Smith, Susan M E; Morgan, Deri; Musset, Boris; Holyoake, John; Chakrabarti, Nilmadhab; Cherny, Vladimir V; DeCoursey, Thomas E; Pomès, Régis

    2013-04-01

    The topological similarity of voltage-gated proton channels (H(V)1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether H(V)1s have a similar structure. We present the construction and validation of a homology model of the human H(V)1 (hH(V)1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hH(V)1 by exploiting the homology of hH(V)1 with VSDs of K(+) and Na(+) channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hH(V)1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp(112). Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hH(V)1. Each Arg residue in the S4 helix of hH(V)1 was replaced by His to test accessibility using Zn(2+) as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg(211), the third Arg residue in the S4 helix in hH(V)1, remains accessible to the internal solution and is located near the

  12. Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

    Energy Technology Data Exchange (ETDEWEB)

    Nakatani, Miyuki [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Ito, Jumpei [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Japan Society for the Promotion of Science, Tokyo, 102-0083 (Japan); Koyama, Riko [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Iijima, Masumi; Yoshimoto, Nobuo [The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047 (Japan); Niimi, Tomoaki [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Kuroda, Shun' ichi [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047 (Japan); Maturana, Andrés D., E-mail: maturana@agr.nagoya-u.ac.jp [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan)

    2016-05-27

    Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.

  13. Sequence-structure relationships in RNA loops: establishing the basis for loop homology modeling.

    Science.gov (United States)

    Schudoma, Christian; May, Patrick; Nikiforova, Viktoria; Walther, Dirk

    2010-01-01

    The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence-structure relationships in loops. Loops differing by structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

  14. Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST

    OpenAIRE

    Goonesekere, Nalin CW

    2009-01-01

    Nalin CW GoonesekereDepartment of Chemistry and Biochemistry, University of Northern iowa, Cedar Falls, IA, USAAbstract: The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution ...

  15. Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model.

    Science.gov (United States)

    Kai, Zhen-Peng; Zhu, Jing-Jing; Deng, Xi-Le; Yang, Xin-Ling; Chen, Shan-Shan

    2018-04-03

    Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT) antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C -terminal amidated octapeptide Manse-AT (6-13). We identified three residues essential for bioactivity (Thr⁴, Arg6 and Phe⁸) by assaying alanine-replacement analogs of Manse-AT (6-13). Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR) was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10-13), we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10-13) validated our hypothesis. The IC 50 value of antagonist Manse-AT (10-13) is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10-13) was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

  16. Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model

    Directory of Open Access Journals (Sweden)

    Zhen-Peng Kai

    2018-04-01

    Full Text Available Insect G protein coupled receptors (GPCRs have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C-terminal amidated octapeptide Manse-AT (6–13. We identified three residues essential for bioactivity (Thr4, Arg6 and Phe8 by assaying alanine-replacement analogs of Manse-AT (6–13. Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10–13, we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10–13 validated our hypothesis. The IC50 value of antagonist Manse-AT (10–13 is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10–13 was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

  17. The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis.

    Science.gov (United States)

    Liou, M L; Liou, H C

    1999-04-09

    The tumor necrosis factor receptor, p60 (TNF-R1), transduces death signals via the association of its cytoplasmic domain with several intracellular proteins. By screening a mammalian cDNA library using the yeast two-hybrid cloning technique, we isolated a ubiquitin-homology protein, DAP-1, which specifically interacts with the cytoplasmic death domain of TNF-R1. Sequence analysis reveals that DAP-1 shares striking sequence homology with the yeast SMT3 protein that is essential for the maintenance of chromosome integrity during mitosis (Meluh, P. B., and Koshland, D. (1995) Mol. Biol. Cell 6, 793-807). DAP-1 is nearly identical to PIC1, a protein that interacts with the PML tumor suppressor implicated in acute promyelocytic leukemia (Boddy, M. N., Howe, K., Etkin, L. D., Solomon, E., and Freemont, P. S. (1996) Oncogene 13, 971-982), and the sentrin protein, which associates with the Fas death receptor (Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M., and Yeh, E. T. (1996) J. Immunol. 157, 4277-4281). The in vivo interaction between DAP-1 and TNF-R1 was further confirmed in mammalian cells. In transient transfection assays, overexpression of DAP-1 suppresses NF-kappaB/Rel activity in 293T cells, a human kidney embryonic carcinoma cell line. Overexpression of either DAP-1 or sentrin causes apoptosis of TNF-sensitive L929 fibroblast cell line, as well as TNF-resistant osteosarcoma cell line, U2OS. Furthermore, the dominant negative Fas-associated death domain protein (FADD) protein blocks the cell death induced by either DAP-1 or FADD. Collectively, these observations highly suggest a role for DAP-1 in mediating TNF-induced cell death signaling pathways, presumably through the recruitment of FADD death effector.

  18. The Mitochondrial DNA (mtDNA)-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination

    KAUST Repository

    Blomme, Jonas

    2017-04-19

    In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gainand loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.

  19. PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

    Science.gov (United States)

    Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

    2016-07-08

    The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Binding site analysis of full-length α1a adrenergic receptor using homology modeling and molecular docking

    International Nuclear Information System (INIS)

    Pedretti, Alessandro; Elena Silva, Maria; Villa, Luigi; Vistoli, Giulio

    2004-01-01

    The recent availability of crystal structure of bovine rhodopsin offers new opportunities in order to approach the construction of G protein coupled receptors. This study focuses the attention on the modeling of full-length α 1a adrenergic receptor (α 1a -AR) due to its biological role and significant implications in pharmacological treatment of benign prostate hyperplasia. This work could be considered made up by two main steps: (a) the construction of full structure of α 1a -AR, through homology modeling methods; (b) the automated docking of an endogenous agonist, norepinephrine, and of an antagonist, WB-4101, using BioDock program. The obtained results highlight the key residues involved in binding sites of both agonists and antagonists, confirming the mutagenesis data and giving new suggestions for the rational design of selective ligands

  1. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

    Science.gov (United States)

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

  2. Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System.

    Science.gov (United States)

    An, Mahru C; O'Brien, Robert N; Zhang, Ningzhe; Patra, Biranchi N; De La Cruz, Michael; Ray, Animesh; Ellerby, Lisa M

    2014-04-15

    We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.

  3. A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities.

    Science.gov (United States)

    Bastien, Olivier; Ortet, Philippe; Roy, Sylvaine; Maréchal, Eric

    2005-03-10

    Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction. We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny. The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations.

  4. A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities

    Directory of Open Access Journals (Sweden)

    Maréchal Eric

    2005-03-01

    Full Text Available Abstract Background Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons and be the basis for a novel method of consistent and stable phylogenetic reconstruction. Results We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny. Conclusion The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations.

  5. Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

    Science.gov (United States)

    Abraham, Anup; Chandler, Douglas E

    2017-10-01

    Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

  6. Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes

    Energy Technology Data Exchange (ETDEWEB)

    Mushegian, Arcady R., E-mail: mushegian2@gmail.com [Division of Molecular and Cellular Biosciences, National Science Foundation, 4201 Wilson Boulevard, Arlington, VA 22230 (United States); Elena, Santiago F., E-mail: sfelena@ibmcp.upv.es [Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, 46022 València (Spain); The Santa Fe Institute, Santa Fe, NM 87501 (United States)

    2015-02-15

    Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, and positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts. - Highlights: • Sequence region shared by plant virus “30K” movement proteins has an all-beta fold. • Most euphyllophyte genomes contain integrated copies of pararetroviruses. • These integrated virus genomes often include intact movement protein genes. • Molecular evidence suggests that these “30K” genes may be selected for function.

  7. Toward the virtual screening of potential drugs in the homology modeled NAD+ dependent DNA ligase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Singh, Vijai; Somvanshi, Pallavi

    2010-02-01

    DNA ligase is an important enzyme and it plays vital role in the replication and repair; also catalyzes nick joining between adjacent bases of DNA. The NAD(+) dependent DNA ligase is selectively present in eubacteria and few viruses; but missing in humans. Homology modeling was used to generate 3-D structure of NAD(+) dependent DNA ligase (LigA) of Mycobacterium tuberculosis using the known template (PDB: 2OWO). Furthermore, the stereochemical quality and torsion angle of 3-D structure was validated. Numerous effective drugs were selected and the active amino acid residue in LigA was targeted and virtual screening through molecular docking was done. In this analysis, four drugs Chloroquine, Hydroxychloroquine, Putrienscine and Adriamycin were found more potent in inhibition of M. tuberculosis through the robust binding affinity between protein-drug interactions in comparison with the other studied drugs. A phylogenetic tree was constructed and it was observed that homology of LigA in M. tuberculosis resembled with other Mycobacterium species. The conserved active amino acids of LigA may be useful to target these drugs. These findings could be used as the starting point of a rational design of novel antibacterial drugs and its analogs.

  8. A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

    DEFF Research Database (Denmark)

    de Vries, R.P.; Burgers, K.; van de Vondervoort, P.J.I

    2004-01-01

    A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces...... very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted....... The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A...

  9. Detecting remote sequence homology in disordered proteins: discovery of conserved motifs in the N-termini of Mononegavirales phosphoproteins.

    Directory of Open Access Journals (Sweden)

    David Karlin

    Full Text Available Paramyxovirinae are a large group of viruses that includes measles virus and parainfluenza viruses. The viral Phosphoprotein (P plays a central role in viral replication. It is composed of a highly variable, disordered N-terminus and a conserved C-terminus. A second viral protein alternatively expressed, the V protein, also contains the N-terminus of P, fused to a zinc finger. We suspected that, despite their high variability, the N-termini of P/V might all be homologous; however, using standard approaches, we could previously identify sequence conservation only in some Paramyxovirinae. We now compared the N-termini using sensitive sequence similarity search programs, able to detect residual similarities unnoticeable by conventional approaches. We discovered that all Paramyxovirinae share a short sequence motif in their first 40 amino acids, which we called soyuz1. Despite its short length (11-16aa, several arguments allow us to conclude that soyuz1 probably evolved by homologous descent, unlike linear motifs. Conservation across such evolutionary distances suggests that soyuz1 plays a crucial role and experimental data suggest that it binds the viral nucleoprotein to prevent its illegitimate self-assembly. In some Paramyxovirinae, the N-terminus of P/V contains a second motif, soyuz2, which might play a role in blocking interferon signaling. Finally, we discovered that the P of related Mononegavirales contain similarly overlooked motifs in their N-termini, and that their C-termini share a previously unnoticed structural similarity suggesting a common origin. Our results suggest several testable hypotheses regarding the replication of Mononegavirales and suggest that disordered regions with little overall sequence similarity, common in viral and eukaryotic proteins, might contain currently overlooked motifs (intermediate in length between linear motifs and disordered domains that could be detected simply by comparing orthologous proteins.

  10. The cohesion protein SOLO associates with SMC1 and is required for synapsis, recombination, homolog bias and cohesion and pairing of centromeres in Drosophila Meiosis.

    Science.gov (United States)

    Yan, Rihui; McKee, Bruce D

    2013-01-01

    Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome

  11. Evolution and virulence contributions of the autotransporter proteins YapJ and YapK of Yersinia pestis CO92 and their homologs in Y. pseudotuberculosis IP32953.

    Science.gov (United States)

    Lenz, Jonathan D; Temple, Brenda R S; Miller, Virginia L

    2012-10-01

    Yersinia pestis, the causative agent of plague, evolved from the gastrointestinal pathogen Yersinia pseudotuberculosis. Both species have numerous type Va autotransporters, most of which appear to be highly conserved. In Y. pestis CO92, the autotransporter genes yapK and yapJ share a high level of sequence identity. By comparing yapK and yapJ to three homologous genes in Y. pseudotuberculosis IP32953 (YPTB0365, YPTB3285, and YPTB3286), we show that yapK is conserved in Y. pseudotuberculosis, while yapJ is unique to Y. pestis. All of these autotransporters exhibit >96% identity in the C terminus of the protein and identities ranging from 58 to 72% in their N termini. By extending this analysis to include homologous sequences from numerous Y. pestis and Y. pseudotuberculosis strains, we determined that these autotransporters cluster into a YapK (YPTB3285) class and a YapJ (YPTB3286) class. The YPTB3286-like gene of most Y. pestis strains appears to be inactivated, perhaps in favor of maintaining yapJ. Since autotransporters are important for virulence in many bacterial pathogens, including Y. pestis, any change in autotransporter content should be considered for its impact on virulence. Using established mouse models of Y. pestis infection, we demonstrated that despite the high level of sequence identity, yapK is distinct from yapJ in its contribution to disseminated Y. pestis infection. In addition, a mutant lacking both of these genes exhibits an additive attenuation, suggesting nonredundant roles for yapJ and yapK in systemic Y. pestis infection. However, the deletion of the homologous genes in Y. pseudotuberculosis does not seem to impact the virulence of this organism in orogastric or systemic infection models.

  12. Structural insights into a high affinity nanobody:antigen complex by homology modelling

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2017-01-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby...... simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for Rgp...

  13. Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex.

    OpenAIRE

    Keshav, K F; Chen, C; Dutta, A

    1995-01-01

    Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-...

  14. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    Energy Technology Data Exchange (ETDEWEB)

    Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Asaoka, Yoichi; Nishina, Hiroshi [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605 (Japan); Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  15. Structural insights into a high affinity nanobody:antigen complex by homology modelling.

    Science.gov (United States)

    Skottrup, Peter Durand

    2017-09-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for RgpB binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified. Collectively, the VHH7 homology model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Variable selection in near infrared spectroscopy for quantitative models of homologous analogs of cephalosporins

    Directory of Open Access Journals (Sweden)

    Yan-Chun Feng

    2014-07-01

    Full Text Available Two universal spectral ranges (4550–4100 cm-1 and 6190–5510 cm-1 for construction of quantitative models of homologous analogs of cephalosporins were proposed by evaluating the performance of five spectral ranges and their combinations, using three data sets of cephalosporins for injection, i.e., cefuroxime sodium, ceftriaxone sodium and cefoperazone sodium. Subsequently, the proposed ranges were validated by using eight calibration sets of other homologous analogs of cephalosporins for injection, namely cefmenoxime hydrochloride, ceftezole sodium, cefmetazole, cefoxitin sodium, cefotaxime sodium, cefradine, cephazolin sodium and ceftizoxime sodium. All the constructed quantitative models for the eight kinds of cephalosporins using these universal ranges could fulfill the requirements for quick quantification. After that, competitive adaptive reweighted sampling (CARS algorithm and infrared (IR–near infrared (NIR two-dimensional (2D correlation spectral analysis were used to determine the scientific basis of these two spectral ranges as the universal regions for the construction of quantitative models of cephalosporins. The CARS algorithm demonstrated that the ranges of 4550–4100 cm-1 and 6190–5510 cm-1 included some key wavenumbers which could be attributed to content changes of cephalosporins. The IR–NIR 2D spectral analysis showed that certain wavenumbers in these two regions have strong correlations to the structures of those cephalosporins that were easy to degrade.

  17. Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

    Science.gov (United States)

    Lu, Y. T.; Hidaka, H.; Feldman, L. J.

    1996-01-01

    Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

  18. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

    Science.gov (United States)

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-08-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

  19. Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids.

    Science.gov (United States)

    Pokhrel, Nilisha; Origanti, Sofia; Davenport, Eric Parker; Gandhi, Disha; Kaniecki, Kyle; Mehl, Ryan A; Greene, Eric C; Dockendorff, Chris; Antony, Edwin

    2017-09-19

    An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs

    International Nuclear Information System (INIS)

    Shanklin, J.; Somerville, C.

    1991-01-01

    Stearoyl-acyl-carrier-protein (ACP) desaturase was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the Δ 9 desaturase is developmentally regulated

  1. Functional equivalency inferred from "authoritative sources" in networks of homologous proteins.

    Science.gov (United States)

    Natarajan, Shreedhar; Jakobsson, Eric

    2009-06-12

    A one-on-one mapping of protein functionality across different species is a critical component of comparative analysis. This paper presents a heuristic algorithm for discovering the Most Likely Functional Counterparts (MoLFunCs) of a protein, based on simple concepts from network theory. A key feature of our algorithm is utilization of the user's knowledge to assign high confidence to selected functional identification. We show use of the algorithm to retrieve functional equivalents for 7 membrane proteins, from an exploration of almost 40 genomes form multiple online resources. We verify the functional equivalency of our dataset through a series of tests that include sequence, structure and function comparisons. Comparison is made to the OMA methodology, which also identifies one-on-one mapping between proteins from different species. Based on that comparison, we believe that incorporation of user's knowledge as a key aspect of the technique adds value to purely statistical formal methods.

  2. Equipment for fully homologous bulb turbine model testing in Laval University

    International Nuclear Information System (INIS)

    Fraser R; Vallée D; Jean Y; Deschênes C

    2014-01-01

    Within the context of liberalisation of the energy market, hydroelectricity remains a first class source of clean and renewable energy. Combining the growing demand of energy, its increasing value and the appreciation associated to the sustainable development, low head sites formerly considered as non-profitable are now exploitable. Bulb turbines likely to equip such sites are traditionally developed on model using right angle transmission leading to piers enlargement for power take off shaft passage, thus restricting possibilities to have fully homologous hydraulic passages. Aiming to sustain good quality development on fully homologous scale model of bulb turbines, the Hydraulic Machines Laboratory (LAMH) of Laval University has developed a brake with an enhanced power to weight ratio. This powerful brake is small enough to be located in the bulb shell while dissipating power without mandatory test head reduction. This paper first presents the basic technology of this brake and its application. Then both its main performance capabilities and dimensional characteristics will be detailed. The instrumentation used to perform accurate measurements will be finally presented

  3. Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-01-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

  4. Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

    DEFF Research Database (Denmark)

    Terp, G E; Christensen, I T; Jørgensen, Flemming Steen

    2000-01-01

    Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach...... to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions...... in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural...

  5. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

    Directory of Open Access Journals (Sweden)

    Eugénie Ansseau

    Full Text Available Hundreds of double homeobox (DUX genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD. In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain and DUX1 (which is limited to the double homeodomain. Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs. Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs

  6. Homologous recombination control by the anti-apoptotic onco-protein Bcl-2

    International Nuclear Information System (INIS)

    Dumay, A.

    2003-12-01

    This research thesis deals with the different biological mechanisms, notably the repair and apoptosis mechanisms induced by irradiation in cells. After a presentation of the genotoxic stress and DNA repair mechanisms, the author discusses the cellular response to a DNA double-strand break, and the regulation of these response mechanisms (how a cellular response emerges: life or death). The next part deals with the apoptosis (cell death by necrosis or apoptosis), and presents the BCL-2 protein family. Results are then reported on laboratory studies of the effect of this protein family

  7. Structure homology and interaction redundancy for discovering virus–host protein interactions

    Science.gov (United States)

    de Chassey, Benoît; Meyniel-Schicklin, Laurène; Aublin-Gex, Anne; Navratil, Vincent; Chantier, Thibaut; André, Patrice; Lotteau, Vincent

    2013-01-01

    Virus–host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high-confidence interactions between viral and human proteins through a combination of structure and high-quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication. PMID:24008843

  8. Structure homology and interaction redundancy for discovering virus-host protein interactions.

    Science.gov (United States)

    de Chassey, Benoît; Meyniel-Schicklin, Laurène; Aublin-Gex, Anne; Navratil, Vincent; Chantier, Thibaut; André, Patrice; Lotteau, Vincent

    2013-10-01

    Virus-host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high-confidence interactions between viral and human proteins through a combination of structure and high-quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication.

  9. Direct association between the Ret receptor tyrosine kinase and the Src homology 2-containing adapter protein Grb7.

    Science.gov (United States)

    Pandey, A; Liu, X; Dixon, J E; Di Fiore, P P; Dixit, V M

    1996-05-03

    Adapter proteins containing Src homology 2 (SH2) domains link transmembrane receptor protein-tyrosine kinases to downstream signal transducing molecules. A family of SH2 containing adapter proteins including Grb7 and Grb10 has been recently identified. We had previously shown that Grb10 associates with Ret via its SH2 domain in an activation-dependent manner (Pandey, A., Duan, H., Di Fiore, P.P., and Dixit, V.M. (1995) J. Biol, Chem. 270, 21461-21463). We now demonstrate that the related adapter molecule Grb7 also associates with Ret in vitro and in vivo, and that the binding of the SH2 domain of Grb7 to Ret is direct. This binding is dependent upon Ret autophosphorylation since Grb7 is incapable of binding a kinase-defective mutant of Ret. Thus two members of the Grb family, Grb7 and Grb10, likely relay signals emanating from Ret to other, as yet, unidentified targets within the cell.

  10. Homology modeling of parasite histone deacetylases to guide the structure-based design of selective inhibitors.

    Science.gov (United States)

    Melesina, Jelena; Robaa, Dina; Pierce, Raymond J; Romier, Christophe; Sippl, Wolfgang

    2015-11-01

    Histone deacetylases (HDACs) are promising epigenetic targets for the treatment of various diseases, including cancer and neurodegenerative disorders. There is evidence that they can also be addressed to treat parasitic infections. Recently, the first X-ray structure of a parasite HDAC was published, Schistosoma mansoni HDAC8, giving structural insights into its inhibition. However, most of the targets from parasites of interest still lack this structural information. Therefore, we prepared homology models of relevant parasitic HDACs and compared them to human and S. mansoni HDACs. The information about known S. mansoni HDAC8 inhibitors and compounds that affect the growth of Trypanosoma, Leishmania and Plasmodium species was used to validate the models by docking and molecular dynamics studies. Our results provide analysis of structural features of parasitic HDACs and should be helpful for selecting promising candidates for biological testing and for structure-based optimisation of parasite-specific inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Preliminary structural studies on the leucine-zipper homology region of the human protein Bap31

    Energy Technology Data Exchange (ETDEWEB)

    Mukasa, Takashi; Santelli, Eugenio [Program on Infectious Diseases, Center for Inflammation and Infectious Diseases, The Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 (United States); Reed, John C. [Program on Apoptosis, Cancer Center, The Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 (United States); Pascual, Jaime, E-mail: pascual@burnham.org [Program on Infectious Diseases, Center for Inflammation and Infectious Diseases, The Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 (United States)

    2007-04-01

    A leucine-zipper with properties as apoptotic regulator in the ER has been crystallized. X-ray data to 2.5 Å resolution were collected, molecular replacement solutions were identified and refinement has been started. B-cell receptor-associated protein 31 (Bap31) is an integral membrane protein located in the endoplasmic reticulum (ER) that participates in the transport and quality control of membrane proteins and plays a role in determining cell sensitivity to ER stress and apoptosis. Its cytoplasmic region contains two target sites for caspase cleavage in certain apoptotic pathways. Here, the subcloning, expression, purification and crystallization of the Homo sapiens Bap31 leucine-zipper C-terminal fragment, which spans residues Gly160–Glu246, are reported. An N-terminally His-tagged protein was overexpressed in Escherichia coli and purified by chromatographic methods. X-ray diffraction data were collected in-house to 2.5 Å resolution. Crystals belong to space group P6{sub 1}22/P6{sub 5}22, with unit-cell parameters a = b = 70.7, c = 80.6 Å. Data analysis indicates the presence of one molecule per asymmetric unit.

  12. Preliminary structural studies on the leucine-zipper homology region of the human protein Bap31

    International Nuclear Information System (INIS)

    Mukasa, Takashi; Santelli, Eugenio; Reed, John C.; Pascual, Jaime

    2007-01-01

    A leucine-zipper with properties as apoptotic regulator in the ER has been crystallized. X-ray data to 2.5 Å resolution were collected, molecular replacement solutions were identified and refinement has been started. B-cell receptor-associated protein 31 (Bap31) is an integral membrane protein located in the endoplasmic reticulum (ER) that participates in the transport and quality control of membrane proteins and plays a role in determining cell sensitivity to ER stress and apoptosis. Its cytoplasmic region contains two target sites for caspase cleavage in certain apoptotic pathways. Here, the subcloning, expression, purification and crystallization of the Homo sapiens Bap31 leucine-zipper C-terminal fragment, which spans residues Gly160–Glu246, are reported. An N-terminally His-tagged protein was overexpressed in Escherichia coli and purified by chromatographic methods. X-ray diffraction data were collected in-house to 2.5 Å resolution. Crystals belong to space group P6 1 22/P6 5 22, with unit-cell parameters a = b = 70.7, c = 80.6 Å. Data analysis indicates the presence of one molecule per asymmetric unit

  13. Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology

    International Nuclear Information System (INIS)

    Shen Yang; Bax, Ad

    2007-01-01

    Chemical shifts of nuclei in or attached to a protein backbone are exquisitely sensitive to their local environment. A computer program, SPARTA, is described that uses this correlation with local structure to predict protein backbone chemical shifts, given an input three-dimensional structure, by searching a newly generated database for triplets of adjacent residues that provide the best match in φ/ψ/χ 1 torsion angles and sequence similarity to the query triplet of interest. The database contains 15 N, 1 H N , 1 H α , 13 C α , 13 C β and 13 C' chemical shifts for 200 proteins for which a high resolution X-ray (≤2.4 A) structure is available. The relative importance of the weighting factors for the φ/ψ/χ 1 angles and sequence similarity was optimized empirically. The weighted, average secondary shifts of the central residues in the 20 best-matching triplets, after inclusion of nearest neighbor, ring current, and hydrogen bonding effects, are used to predict chemical shifts for the protein of known structure. Validation shows good agreement between the SPARTA-predicted and experimental shifts, with standard deviations of 2.52, 0.51, 0.27, 0.98, 1.07 and 1.08 ppm for 15 N, 1 H N , 1 H α , 13 C α , 13 C β and 13 C', respectively, including outliers

  14. Novel features of a PIWI-like protein homolog in the parasitic protozoan Leishmania.

    Directory of Open Access Journals (Sweden)

    Prasad K Padmanabhan

    Full Text Available In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI((-/- mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

  15. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

    OpenAIRE

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-01-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only...

  16. In silico predictive studies of mAHR congener binding using homology modelling and molecular docking.

    Science.gov (United States)

    Panda, Roshni; Cleave, A Suneetha Susan; Suresh, P K

    2014-09-01

    The aryl hydrocarbon receptor (AHR) is one of the principal xenobiotic, nuclear receptor that is responsible for the early events involved in the transcription of a complex set of genes comprising the CYP450 gene family. In the present computational study, homology modelling and molecular docking were carried out with the objective of predicting the relationship between the binding efficiency and the lipophilicity of different polychlorinated biphenyl (PCB) congeners and the AHR in silico. Homology model of the murine AHR was constructed by several automated servers and assessed by PROCHECK, ERRAT, VERIFY3D and WHAT IF. The resulting model of the AHR by MODWEB was used to carry out molecular docking of 36 PCB congeners using PatchDock server. The lipophilicity of the congeners was predicted using the XLOGP3 tool. The results suggest that the lipophilicity influences binding energy scores and is positively correlated with the same. Score and Log P were correlated with r = +0.506 at p = 0.01 level. In addition, the number of chlorine (Cl) atoms and Log P were highly correlated with r = +0.900 at p = 0.01 level. The number of Cl atoms and scores also showed a moderate positive correlation of r = +0.481 at p = 0.01 level. To the best of our knowledge, this is the first study employing PatchDock in the docking of AHR to the environmentally deleterious congeners and attempting to correlate structural features of the AHR with its biochemical properties with regards to PCBs. The result of this study are consistent with those of other computational studies reported in the previous literature that suggests that a combination of docking, scoring and ranking organic pollutants could be a possible predictive tool for investigating ligand-mediated toxicity, for their subsequent validation using wet lab-based studies. © The Author(s) 2012.

  17. SCRINING IN SILICO ACTIVE COMPOUND OF Pachyrrhizus erosus AS ANTITIROSINASE ON Aspergillus oryzae (COMPUTATTIONAL STUDY WITH HOMOLOGY MODELING AND MOLECULAR DOCKING

    Directory of Open Access Journals (Sweden)

    Endang Lukitaningsih

    2015-11-01

    Full Text Available Bengkoang telah banyak digunakan dalam industri kosmetika sebagai whitening agent. Berdasarkan penelitian Lukitaningsih (2009, bengkoang mengandung 6 senyawa aktif yang mampu berperan sebagai whitening agent dengan menghambat aktivitas enzim tirosinase dari jamur Aspergillus oryzae (TyrAo. Namun interaksi senyawa aktif bengkoang dalam menghambat enzim tirosinase belum dapat diketahui. Interaksi senyawa-senyawa aktif bengkoang dengan enzim TyrAo dapat diketahui dengan studi komputasional (in silico. Pemodelan interaksi senyawa aktif bengkoang dengan enzim TyrAo dilakukan dengan metode homology modeling dan molecular docking. Homology modeling dilakukan untuk memodelkan struktur tiga dimensi (3D enzim tirosinase Aspergillus oryzae (TyrAo melalui template berupa protein homolog yang sudah diketahui struktur 3D-nya yaitu enzim TyrAb (PDBID: 2Y9X. Model TyrAo digunakan sebagai target makromolekul dalam metode molecular docking. Metode molecular docking merupakan metode untuk menggambarkan posisi ligan (senyawa-senyawa aktif bengkoang pada sisi aktif reseptor (model TyrAo. Berdasarkan docking yang dilakukan diketahui bahwa residu-residu yang banyak berpengaruh pada interaksi ligan pada sisi aktif adalah residu Thr275 yang berinteraksi secara ikatan hidrogen dengan ligan dan residu His294 yang berinteraksi secara hidrofobik pada cincin aromatik ligan. Penelitian in silico dan in vitro yang telah dilakukan memiliki korelasi (R2 sebesar -0,8366. Korelasi ini menandakan bahwa aktivitas senyawa-senyawa aktif pada bengkoang dalam menghambat enzim TyrAo memiliki hasil yang serupa pada penelitian yang  dilakukan secara in silico dan in vitro.

  18. Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.

    Science.gov (United States)

    Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I

    1988-11-25

    . Finally, neither all-trans-retinol nor retinoic acid bound to E. coli-derived rat intestinal fatty acid-binding protein, a homologous protein whose tertiary structure is known. Together, the data suggest that these three family members have acquired unique functional capabilities.

  19. Identification of three homologous latex-clearing protein (lcp) genes from the genome of Streptomyces sp. strain CFMR 7.

    Science.gov (United States)

    Nanthini, Jayaram; Ong, Su Yean; Sudesh, Kumar

    2017-09-10

    Rubber materials have greatly contributed to human civilization. However, being a polymeric material does not decompose easily, it has caused huge environmental problems. On the other hand, only few bacteria are known to degrade rubber, with studies pertaining them being intensively focusing on the mechanism involved in microbial rubber degradation. The Streptomyces sp. strain CFMR 7, which was previously confirmed to possess rubber-degrading ability, was subjected to whole genome sequencing using the single molecule sequencing technology of the PacBio® RS II system. The genome was further analyzed and compared with previously reported rubber-degrading bacteria in order to identify the potential genes involved in rubber degradation. This led to the interesting discovery of three homologues of latex-clearing protein (Lcp) on the chromosome of this strain, which are probably responsible for rubber degrading activities. Genes encoding oxidoreductase α-subunit (oxiA) and oxidoreductase β-subunit (oxiB) were also found downstream of two lcp genes which are located adjacent to each other. In silico analysis reveals genes that have been identified to be involved in the microbial degradation of rubber in the Streptomyces sp. strain CFMR 7. This is the first whole genome sequence of a clear-zone-forming natural rubber- degrading Streptomyces sp., which harbours three Lcp homologous genes with the presence of oxiA and oxiB genes compared to the previously reported Gordonia polyisoprenivorans strain VH2 (with two Lcp homologous genes) and Nocardia nova SH22a (with only one Lcp gene). Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Wetsel, R A; Tack, B F

    1986-01-01

    We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide...... sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had...... a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences...

  1. GmGBP1, a homolog of human ski interacting protein in soybean, regulates flowering and stress tolerance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Zhang Yanwei

    2013-02-01

    Full Text Available Abstract Background SKIP is a transcription cofactor in many eukaryotes. It can regulate plant stress tolerance in rice and Arabidopsis. But the homolog of SKIP protein in soybean has been not reported up to now. Results In this study, the expression patterns of soybean GAMYB binding protein gene (GmGBP1 encoding a homolog of SKIP protein were analyzed in soybean under abiotic stresses and different day lengths. The expression of GmGBP1 was induced by polyethyleneglycol 6000, NaCl, gibberellin, abscisic acid and heat stress. GmGBP1 had transcriptional activity in C-terminal. GmGBP1 could interact with R2R3 domain of GmGAMYB1 in SKIP domain to take part in gibberellin flowering pathway. In long-day (16 h-light condition, transgenic Arabidopsis with the ectopic overexpression of GmGBP1 exhibited earlier flowering and less number of rosette leaves; Suppression of AtSKIP in Arabidopsis resulted in growth arrest, flowering delay and down-regulation of many flowering-related genes (CONSTANS, FLOWERING LOCUS T, LEAFY; Arabidopsis myb33 mutant plants with ectopic overexpression of GmGBP1 showed the same flowering phenotype with wild type. In short-day (8 h-light condition, transgenic Arabidopsis plants with GmGBP1 flowered later and showed a higher level of FLOWERING LOCUS C compared with wild type. When treated with abiotic stresses, transgenic Arabidopsis with the ectopic overexpression of GmGBP1 enhanced the tolerances to heat and drought stresses but reduced the tolerance to high salinity, and affected the expressions of several stress-related genes. Conclusions In Arabidopsis, GmGBP1 might positively regulate the flowering time by affecting CONSTANS, FLOWERING LOCUS T, LEAFY and GAMYB directly or indirectly in photoperiodic and gibberellin pathways in LDs, but GmGBP1 might represse flowering by affecting FLOWERING LOCUS C and SHORT VEGETATIVE PHASE in autonomous pathway in SDs. GmGBP1 might regulate the activity of ROS-eliminating to improve the

  2. Classical Bovine Spongiform Encephalopathy by Transmission of H-Type Prion in Homologous Prion Protein Context

    OpenAIRE

    Torres, Juan-María; Andréoletti, Olivier; Lacroux, Caroline; Prieto, Irene; Lorenzo, Patricia; Larska, Magdalena; Baron, Thierry; Espinosa, Juan-Carlos

    2011-01-01

    Bovine spongiform encephalopathy (BSE) and BSErelated disorders have been associated with a single major prion strain. Recently, 2 atypical, presumably sporadic forms of BSE have been associated with 2 distinct prion strains that are characterized mainly by distinct Western blot profi les of abnormal protease-resistant prion protein (PrPres), named high-type (BSE-H) and low-type (BSE-L), that also differed from classical BSE. We characterized 5 atypical BSE-H isolates by analyzing their molec...

  3. The Fanconi anemia group A protein modulates homologous repair of DNA double-strand breaks in mammalian cells.

    Science.gov (United States)

    Yang, Yun-Gui; Herceg, Zdenko; Nakanishi, Koji; Demuth, Ilja; Piccoli, Colette; Michelon, Jocelyne; Hildebrand, Gabriele; Jasin, Maria; Digweed, Martin; Wang, Zhao-Qi

    2005-10-01

    Fanconi anemia (FA) cells exhibit hypersensitivity to DNA interstrand cross-links (ICLs) and high levels of chromosome instability. FA gene products have been shown to functionally or physically interact with BRCA1, RAD51 and the MRE11/RAD50/NBS1 complex, suggesting that the FA complex may be involved in the repair of DNA double-strand breaks (DSBs). Here, we have investigated specifically the function of the FA group A protein (FANCA) in the repair of DSBs in mammalian cells. We show that the targeted deletion of Fanca exons 37-39 generates a null for Fanca in mice and abolishes ubiquitination of Fancd2, the downstream effector of the FA complex. Cells lacking Fanca exhibit increased chromosomal aberrations and attenuated accumulation of Brca1 and Rad51 foci in response to DNA damage. The absence of Fanca greatly reduces gene-targeting efficiency in mouse embryonic stem (ES) cells and compromises the survival of fibroblast cells in response to ICL agent treatment. Fanca-null cells exhibit compromised homology-directed repair (HDR) of DSBs, particularly affecting the single-strand annealing pathway. These data identify the Fanca protein as an integral component in the early step of HDR of DSBs and thereby minimizing the genomic instability.

  4. Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

    Science.gov (United States)

    Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

    1998-08-01

    In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

  5. A Temperature-Independent Cold-Shock Protein Homolog Acts as a Virulence Factor in Xylella fastidiosa.

    Science.gov (United States)

    Burbank, Lindsey P; Stenger, Drake C

    2016-05-01

    Xylella fastidiosa, causal agent of Pierce's disease (PD) of grapevine, is a fastidious organism that requires very specific conditions for replication and plant colonization. Cold temperatures reduce growth and survival of X. fastidiosa both in vitro and in planta. However, little is known regarding physiological responses of X. fastidiosa to temperature changes. Cold-shock proteins (CSP), a family of nucleic acid-binding proteins, act as chaperones facilitating translation at low temperatures. Bacterial genomes often encode multiple CSP, some of which are strongly induced following exposure to cold. Additionally, CSP contribute to the general stress response through mRNA stabilization and posttranscriptional regulation. A putative CSP homolog (Csp1) with RNA-binding activity was identified in X. fastidiosa Stag's Leap. The csp1 gene lacked the long 5' untranslated region characteristic of cold-inducible genes and was expressed in a temperature-independent manner. As compared with the wild type, a deletion mutant of csp1 (∆csp1) had decreased survival rates following cold exposure and salt stress in vitro. The deletion mutant also was significantly less virulent in grapevine, as compared with the wild type, in the absence of cold stress. These results suggest an important function of X. fastidiosa Csp1 in response to cellular stress and during plant colonization.

  6. Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection

    Science.gov (United States)

    Tran, Si C.; Pham, Tu M.; Nguyen, Lam N.; Park, Eun-Mee; Lim, Yun-Sook

    2016-01-01

    ABSTRACT Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCE TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of

  7. NGF-Dependent neurite outgrowth in PC12 cells overexpressing the Src homology 2-domain protein shb requires activation of the Rap1 pathway

    NARCIS (Netherlands)

    Lu, L.; Annerén, C.; Reedquist, K. A.; Bos, J. L.; Welsh, M.

    2000-01-01

    The Src homology 2 (SH2) domain adaptor protein Shb has been shown to transmit NGF- and FGF-2-dependent differentiation signals in PC12 cells. To study if this involves signaling through the small GTPase Rap1, Rap1 activity was assessed in Shb-overexpressing PC12 cells. We demonstrate that NGF and

  8. The Protein Model Portal--a comprehensive resource for protein structure and model information.

    Science.gov (United States)

    Haas, Juergen; Roth, Steven; Arnold, Konstantin; Kiefer, Florian; Schmidt, Tobias; Bordoli, Lorenza; Schwede, Torsten

    2013-01-01

    The Protein Model Portal (PMP) has been developed to foster effective use of 3D molecular models in biomedical research by providing convenient and comprehensive access to structural information for proteins. Both experimental structures and theoretical models for a given protein can be searched simultaneously and analyzed for structural variability. By providing a comprehensive view on structural information, PMP offers the opportunity to apply consistent assessment and validation criteria to the complete set of structural models available for proteins. PMP is an open project so that new methods developed by the community can contribute to PMP, for example, new modeling servers for creating homology models and model quality estimation servers for model validation. The accuracy of participating modeling servers is continuously evaluated by the Continuous Automated Model EvaluatiOn (CAMEO) project. The PMP offers a unique interface to visualize structural coverage of a protein combining both theoretical models and experimental structures, allowing straightforward assessment of the model quality and hence their utility. The portal is updated regularly and actively developed to include latest methods in the field of computational structural biology. Database URL: http://www.proteinmodelportal.org.

  9. The Protein Model Portal—a comprehensive resource for protein structure and model information

    Science.gov (United States)

    Haas, Juergen; Roth, Steven; Arnold, Konstantin; Kiefer, Florian; Schmidt, Tobias; Bordoli, Lorenza; Schwede, Torsten

    2013-01-01

    The Protein Model Portal (PMP) has been developed to foster effective use of 3D molecular models in biomedical research by providing convenient and comprehensive access to structural information for proteins. Both experimental structures and theoretical models for a given protein can be searched simultaneously and analyzed for structural variability. By providing a comprehensive view on structural information, PMP offers the opportunity to apply consistent assessment and validation criteria to the complete set of structural models available for proteins. PMP is an open project so that new methods developed by the community can contribute to PMP, for example, new modeling servers for creating homology models and model quality estimation servers for model validation. The accuracy of participating modeling servers is continuously evaluated by the Continuous Automated Model EvaluatiOn (CAMEO) project. The PMP offers a unique interface to visualize structural coverage of a protein combining both theoretical models and experimental structures, allowing straightforward assessment of the model quality and hence their utility. The portal is updated regularly and actively developed to include latest methods in the field of computational structural biology. Database URL: http://www.proteinmodelportal.org PMID:23624946

  10. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

    Science.gov (United States)

    Kadamur, Ganesh

    2016-01-01

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

  11. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

    Science.gov (United States)

    Kadamur, Ganesh; Ross, Elliott M

    2016-05-20

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

    Science.gov (United States)

    Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Modeling Mercury in Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jeremy C [ORNL; Parks, Jerry M [ORNL

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

  14. Homology modeling of dissimilatory APS reductases (AprBA of sulfur-oxidizing and sulfate-reducing prokaryotes.

    Directory of Open Access Journals (Sweden)

    Birte Meyer

    Full Text Available BACKGROUND: The dissimilatory adenosine-5'-phosphosulfate (APS reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP; in sulfur-oxidizing bacteria (SOB it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle. METHODOLOGY/PRINCIPAL FINDINGS: Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8-99.3% and 89.5-96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 A with respect to the template structures. Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1 SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2 crenarchaeal SRP Caldivirga and Pyrobaculum, and (3 SOB of the distinct Apr lineage I and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from. CONCLUSIONS: Based on the comparative

  15. Schizosaccharomyces pombe Homologs of Human DJ-1 Are Stationary Phase-Associated Proteins That Are Involved in Autophagy and Oxidative Stress Resistance.

    Directory of Open Access Journals (Sweden)

    Yang Su

    Full Text Available The Parkinson's disease protein DJ-1 is involved in various cellular functions including detoxification of dicarbonyl compounds, autophagy and oxidative stress response. DJ-1 homologs are widely found in both prokaryotes and eukaryotes, constituting a superfamily of proteins that appear to be involved in stress response. Schizosaccharomyces pombe contains six DJ-1 homologs, designated Hsp3101-Hsp3105 and Sdj1 (previously named SpDJ-1. Here we show that deletion of any one of these six genes somehow affects autophagy during prolonged stationary phase. Furthermore, deletions of each of these DJ-1 homologs result in reduced stationary phase survival. Deletion of sdj1 also increases the sensitivity of stationary-phase cells to oxidative stress induced by hydrogen peroxide (H2O2 whereas overexpression of sdj1 has the opposite effect. Consistent with their role in stationary phase, expression of hsp3101, hsp3102, hsp3105 and sdj1, and to a lesser extent hsp3103 and hsp3104, is increased in stationary phase. The induction of hsp3101, hsp3102, hsp3105 and sdj1 involves the Sty1-regulated transcription factor Atf1 but not the transcription factor Pap1. Our results firmly establish that S. pombe homologs of DJ-1 are stationary-phase associated proteins and are likely involved in autophagy and antioxidant defense in stationary phase of S. pombe cells.

  16. HOMOLOGY MODELING AND FUNCTIONAL CHARACTERIZATION OF THREE-DIMENSIONAL STRUCTURE OF DAHP SYNTHASE FROM BRACHYPODIUM DISTACHYON

    Directory of Open Access Journals (Sweden)

    Aditya Dev

    2013-06-01

    Full Text Available The Shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in microbes and plants but absent in animals. The 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS is the first enzyme of this pathway, which is involved in the condensation of phosphoenolpyruvate (PEP and D-erythrose 4-phosphate (E4P to produce 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP. DAHPS enzymes have been divided into two types, class I and class II, based on their primary amino acid sequence and three dimensional structures. The plant DAHPS belongs to class II and is regulated differently than DAHPS from microorganisms. To understand the structural basis of such differences in DAHPS from plants and its catalytic mechanism, we have used sequence analysis, homology modeling and docking approach to generate the three dimensional models of DAHP synthase from Brachypodium distachyon (Bd-DAHPS complexed with substrate PEP for the first time. The three dimensional models of Bd-DAHPS provides a detailed knowledge of the active site and the important secondary structural regions that play significant roles in the regulatory mechanism and further may be helpful for design of specific inhibitors towards herbicide development.

  17. Top-Down-Assisted Bottom-Up Method for Homologous Protein Sequencing: Hemoglobin from 33 Bird Species

    Science.gov (United States)

    Song, Yang; Laskay, Ünige A.; Vilcins, Inger-Marie E.; Barbour, Alan G.; Wysocki, Vicki H.

    2015-11-01

    Ticks are vectors for disease transmission because they are indiscriminant in their feeding on multiple vertebrate hosts, transmitting pathogens between their hosts. Identifying the hosts on which ticks have fed is important for disease prevention and intervention. We have previously shown that hemoglobin (Hb) remnants from a host on which a tick fed can be used to reveal the host's identity. For the present research, blood was collected from 33 bird species that are common in the U.S. as hosts for ticks but that have unknown Hb sequences. A top-down-assisted bottom-up mass spectrometry approach with a customized searching database, based on variability in known bird hemoglobin sequences, has been devised to facilitate fast and complete sequencing of hemoglobin from birds with unknown sequences. These hemoglobin sequences will be added to a hemoglobin database and used for tick host identification. The general approach has the potential to sequence any set of homologous proteins completely in a rapid manner.

  18. Structural insights into transient receptor potential vanilloid type 1 (TRPV1) from homology modeling, flexible docking, and mutational studies.

    Science.gov (United States)

    Lee, Jin Hee; Lee, Yoonji; Ryu, HyungChul; Kang, Dong Wook; Lee, Jeewoo; Lazar, Jozsef; Pearce, Larry V; Pavlyukovets, Vladimir A; Blumberg, Peter M; Choi, Sun

    2011-04-01

    The transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel composed of four monomers with six transmembrane helices (TM1-TM6). TRPV1 is found in the central and peripheral nervous system, and it is an important therapeutic target for pain relief. We describe here the construction of a tetrameric homology model of rat TRPV1 (rTRPV1). We experimentally evaluated by mutational analysis the contribution of residues of rTRPV1 contributing to ligand binding by the prototypical TRPV1 agonists, capsaicin and resiniferatoxin (RTX). We then performed docking analysis using our homology model. The docking results with capsaicin and RTX showed that our homology model was reliable, affording good agreement with our mutation data. Additionally, the binding mode of a simplified RTX (sRTX) ligand as predicted by the modeling agreed well with those of capsaicin and RTX, accounting for the high binding affinity of the sRTX ligand for TRPV1. Through the homology modeling, docking and mutational studies, we obtained important insights into the ligand-receptor interactions at the molecular level which should prove of value in the design of novel TRPV1 ligands.

  19. Roles of Rad51 protein in homologous recombination in mammalian cells: relation with repair, replication and cell cycle

    International Nuclear Information System (INIS)

    Lambert, S.

    2001-01-01

    Homologous recombination (HR) is a fundamental process, allowing a faithful repair. In mammalian, MmRAD51, which is the homologue of Saccharomyces cerevisiae ScRAD51 key protein for HR, is an essential gene. This work is based on the characterisation of viable hyper and hypo-recombinant cell lines specifically affected in the Rad51 pathway. By expressing wild type and dominant negative forms of MmRad51, we demonstrated that Rad51 pathway participates to the repair by HR to induced DNA damages. However, inhibition of the Rad 51 pathway does not affect cell viability, spontaneously or after irradiation, whereas, radiation induced HR is inhibited. In the presence of DNA damages during late S and G2/M phase, inhibition of Rad51 pathway induced chromosomal aberrations, leading to a transient arrest in mitosis. This arrest is associated with an increased of cell death. However, a fraction of cells can escape from this transient arrest by forming tetraploid cells, associated with an absence of chromalid separation. Thus, in response to impaired Rad51 pathway, mitotic checkpoints seems to play an essential role. In line with this, we showed that the essential function of Rad51 is p53-dependent, which is in agreement with the role of p53 in tetraploidy inhibition. Our results suggest that the Rad51 protein could participate to the control of mitotic checkpoints and thus to the maintenance of genetic stability. This function could involve other Rad51 partners such as the tumour suppressors BRCA1, BRCA2 and p53. (author) [fr

  20. Mmu-miR-615-3p regulates lipoapoptosis by inhibiting C/EBP homologous protein.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Miyamoto

    Full Text Available Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP. Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p. We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and in silico analyses, respectively. There is a single miR-615-3p binding site in the 3'untranslated region (UTR of the Chop transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.

  1. Diverse binding site structures revealed in homology models of polyreactive immunoglobulins

    Science.gov (United States)

    Ramsland, Paul A.; Guddat, Luke W.; Edmundson, Allen B.; Raison, Robert L.

    1997-09-01

    We describe here computer-assisted homology models of the combiningsite structure of three polyreactive immunoglobulins. Template-based modelsof Fv (VL-VH) fragments were derived forthe surface IgM expressed by the malignant CD5 positive B cells from threepatients with chronic lymphocytic leukaemia (CLL). The conserved frameworkregions were constructed using crystal coordinates taken from highlyhomologous human variable domain structures (Pot and Hil). Complementaritydetermining regions (CDRs) were predicted by grafting loops, taken fromknown immunoglobulin structures, onto the Fv framework models. The CDRtemplates were chosen, where possible, to be of the same length and of highresidue identity or similarity. LCDR1, 2 and 3 as well as HCDR1 and 2 forthe Fv were constructed using this strategy. For HCDR3 prediction, adatabase containing the Cartesian coordinates of 30 of these loops wascompiled from unliganded antibody X-ray crystallographic structures and anHCDR3 of the same length as that of the B CLL Fv was selected as a template.In one case (Yar), the resulting HCDR3 model gave unfavourable interactionswhen incorporated into the Fv model. This HCDR3 was therefore modelled usingan alternative strategy of construction of the loop stems, using apreviously described HCDR3 conformation (Pot), followed by chain closurewith a β-turn. The template models were subjected to positionalrefinement using energy minimisation and molecular dynamics simulations(X-PLOR). An electrostatic surface description (GRASP) did not reveal acommon structural feature within the binding sites of the three polyreactiveFv. Thus, polyreactive immunoglobulins may recognise similar and multipleantigens through a diverse array of binding site structures.

  2. INHIBITION OF THE DNA-BINDING ACTIVITY OF DROSOPHILA SUPPRESSOR OF HAIRLESS AND OF ITS HUMAN HOMOLOG, KBF2/RBP-J-KAPPA, BY DIRECT PROTEIN-PROTEIN INTERACTION WITH DROSOPHILA HAIRLESS

    NARCIS (Netherlands)

    BROU, C; LOGEAT, F; LECOURTOIS, M; VANDEKERCKHOVE, Joël; KOURILSKY, P; SCHWEISGUTH, F; ISRAEL, A

    1994-01-01

    We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless [Su(H)] gene. Deletion studies of the RBP-J kappa and Su(H) proteins allowed

  3. Tumor necrosis factor alpha of teleosts: in silico characterization and homology modeling

    Directory of Open Access Journals (Sweden)

    Tran Ngoc Tuan

    2016-10-01

    Full Text Available Tumor necrosis factor alpha (TNF- is known to be crucial in many biological activities of organisms. In this study, physicochemical properties and modeling of TNF- protein of fish was analyzed using in silico approach. TNF- proteins selected from fish species, including grass carp (Ctenopharyngodon idella, zebra fish (Danio rerio, Nile tilapia (Oreochromis niloticus, goldfish (Carassius auratus, and rainbow trout (Oncorhynchus mykiss were used in this study. Physicochemical characteristics with molecular weight, theoretical isoelectric point, extinction coefficient, aliphatic index, instability index, total number of negatively charged residues and positively charged residues, and grand average of hydropathicity were computed. All proteins were classified as transmembrane proteins. The “transmembrane region” and “TNF” domain were identified from protein sequences. The function prediction of proteins was also performed. Alpha helices and random coils were dominating in the secondary structure of the proteins. Three-dimensional structures were predicted and verified as good structures for the investigation of TNF- of fish by online server validation.

  4. DNA double strand break repair in mammalian cells: role of MRE11 and BLM proteins at the initiation of Non Homologous End Joining (NHEJ)

    International Nuclear Information System (INIS)

    Grabarz, Anastazja

    2011-01-01

    DNA double strand breaks (DSBs) are highly cytotoxic lesions, which can lead to genetic rearrangements. Two pathways are responsible for repairing these lesions: homologous recombination (HR) and non homologous end joining (NHEJ). In our laboratory, an intrachromosomal substrate has been established in order to measure the efficiency and the fidelity of NHEJ in living cells (Guirouilh-Barbat 2004). This approach led us to identify a KU-independent alternative pathway, which uses micro homologies in the proximity of the junction to accomplish repair - the alternative NHEJ (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). The goal of my thesis consisted in identifying and characterising major actors of this pathway. In the absence of KU, alternative NHEJ would be initiated by ssDNA resection of damaged ends. We showed that the nuclease activity of MRE11 is necessary for this mechanism. MRE11 overexpression leads to a two fold stimulation of NHEJ efficiency, while the extinction of MRE11 by siRNA results in a two fold decrease. Our results demonstrate that the proteins RAD50 and CtIP act in the same pathway as MRE11. Moreover, in cells deficient for XRCC4, MIRIN - an inhibitor of the MRN complex - leads to a decrease in repair efficiency, implicating MRE11 in alternative NHEJ. We also showed that MRE11 can act in an ATM-dependent and independent manner (Rass et Grabarz Nat Struct Mol Biol 2009). The initiation of break resection needs to be pursued by a more extensive degradation of DNA, which is accomplished in yeast by the proteins Exo1 and Sgs1/Dna2. In human cells, in vitro studies have recently proposed a similar model of a two-step break resection. We chose to elucidate the role of one of the human homologs of Sgs1 - the RecQ helicase BLM - in the resection process. Our experiments show, that he absence of BLM decreases the efficiency of end joining by NHEJ, accompanied by an increase in error-prone events, especially long-range deletions (≥200 nt). This

  5. Reaction mechanism of sterol hydroxylation by steroid C25 dehydrogenase - Homology model, reactivity and isoenzymatic diversity.

    Science.gov (United States)

    Rugor, Agnieszka; Wójcik-Augustyn, Anna; Niedzialkowska, Ewa; Mordalski, Stefan; Staroń, Jakub; Bojarski, Andrzej; Szaleniec, Maciej

    2017-08-01

    Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O 2 -independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[ 2 H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Solution structure of tRNA{sup Val} from refinement of homology model against residual dipolar coupling and SAXS data

    Energy Technology Data Exchange (ETDEWEB)

    Grishaev, Alexander, E-mail: AlexanderG@intra.niddk.nih.gov; Ying, Jinfa [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Canny, Marella D.; Pardi, Arthur [University of Colorado, Boulder, Department of Chemistry and Biochemistry, 215 UCB (United States)], E-mail: Arthur.Pardi@Colorado.edu; Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)], E-mail: bax@nih.gov

    2008-10-15

    A procedure is presented for refinement of a homology model of E. coli tRNA{sup Val}, originally based on the X-ray structure of yeast tRNA{sup Phe}, using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N-H{sup N} RDCs measured with Pf1 phage alignment, and 20 imino N-H{sup N} RDCs obtained from magnetic field dependent alignment of tRNA{sup Val}. The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA{sup Phe} by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q{sub free} = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA{sup Phe}, in agreement with previous NMR-based tRNA{sup Val} models.

  7. Homology modeling, molecular dynamics and inhibitor binding study on MurD ligase of Mycobacterium tuberculosis.

    Science.gov (United States)

    Arvind, Akanksha; Kumar, Vivek; Saravanan, Parameswaran; Mohan, C Gopi

    2012-09-01

    The cell wall of mycobacterium offers well validated targets which can be exploited for discovery of new lead compounds. MurC-MurF ligases catalyze a series of irreversible steps in the biosynthesis of peptidoglycan precursor, i.e. MurD catalyzes the ligation of D-glutamate to the nucleotide precursor UMA. The three dimensional structure of Mtb-MurD is not known and was predicted by us for the first time using comparative homology modeling technique. The accuracy and stability of the predicted Mtb-MurD structure was validated using Procheck and molecular dynamics simulation. Key interactions in Mtb-MurD were studied using docking analysis of available transition state inhibitors of E.coli-MurD. The docking analysis revealed that analogues of both L and D forms of glutamic acid have similar interaction profiles with Mtb-MurD. Further, residues His192, Arg382, Ser463, and Tyr470 are proposed to be important for inhibitor-(Mtb-MurD) interactions. We also identified few pharmacophoric features essential for Mtb-MurD ligase inhibitory activity and which can further been utilized for the discovery of putative antitubercular chemotherapy.

  8. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

    OpenAIRE

    Yoga, Yano M. K.; Traore, Daouda A. K.; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R.; Barker, Andrew; Leedman, Peter J.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

    2012-01-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA...

  9. Modeling protein structures: construction and their applications.

    Science.gov (United States)

    Ring, C S; Cohen, F E

    1993-06-01

    Although no general solution to the protein folding problem exists, the three-dimensional structures of proteins are being successfully predicted when experimentally derived constraints are used in conjunction with heuristic methods. In the case of interleukin-4, mutagenesis data and CD spectroscopy were instrumental in the accurate assignment of secondary structure. In addition, the tertiary structure was highly constrained by six cysteines separated by many residues that formed three disulfide bridges. Although the correct structure was a member of a short list of plausible structures, the "best" structure was the topological enantiomer of the experimentally determined conformation. For many proteases, other experimentally derived structures can be used as templates to identify the secondary structure elements. In a procedure called modeling by homology, the structure of a known protein is used as a scaffold to predict the structure of another related protein. This method has been used to model a serine and a cysteine protease that are important in the schistosome and malarial life cycles, respectively. The model structures were then used to identify putative small molecule enzyme inhibitors computationally. Experiments confirm that some of these nonpeptidic compounds are active at concentrations of less than 10 microM.

  10. Generation of a homology model of the human histamine H3 receptor for ligand docking and pharmacophore-based screening

    Science.gov (United States)

    Schlegel, Birgit; Laggner, Christian; Meier, Rene; Langer, Thierry; Schnell, David; Seifert, Roland; Stark, Holger; Höltje, Hans-Dieter; Sippl, Wolfgang

    2007-08-01

    The human histamine H3 receptor (hH3R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH3R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH3R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH3R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH3R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [3H]Nα-methylhistamine binding assay. The compounds tested showed affinities at hH3R with K i values ranging from 0.079 to 6.3 μM.

  11. A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities

    OpenAIRE

    Maréchal Eric; Ortet Philippe; Roy Sylvaine; Bastien Olivier

    2005-01-01

    Abstract Background Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic recon...

  12. Yes-associated protein homolog, YAP-1, is involved in the thermotolerance and aging in the nematode Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Iwasa, Hiroaki [Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519 (Japan); Maimaiti, Sainawaer [Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519 (Japan); Department of Psychotherapy, The Fourth People' s Hospital of Urumqi, Urumqi 830000 (China); Kuroyanagi, Hidehito [Laboratory of Gene Expression, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo 113-8519 (Japan); Kawano, Shodai; Inami, Kazutoshi; Timalsina, Shikshya; Ikeda, Mitsunobu; Nakagawa, Kentaro [Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519 (Japan); Hata, Yutaka, E-mail: yuhammch@tmd.ac.jp [Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519 (Japan)

    2013-04-15

    The mammalian Hippo pathway comprises mammalian Ste20-like kinases (MST1/2) and large tumor suppressor kinases (LATS1/2). LATS1/2, which are activated by MST1/2, phosphorylate a transcriptional co-activator, yes-associated protein (YAP), and induce the recruitment of YAP by 14-3-3 to cytoplasm, so that the TEAD-dependent gene transcriptions are turned off. Although the core components of the Hippo pathway are well conserved in metazoans, it has been discussed that Caenorhabditis elegans lacks YAP ortholog, we found that F13E6.4 gene encodes a protein that shows sequence similarities to YAP in the N-terminal TEAD-binding domain and in the WW domain. We designated this gene as yap-1. YAP-1 is widely expressed in various cells such as epithelial cells, muscles, hypodermal cells, gonadal sheath cells, spermatheca, and hypodermal cells. YAP-1 is distributed in cytoplasm and nuclei. wts-1 (LATS ortholog) and ftt-2 (14-3-3 ortholog) knockdowns cause nuclear accumulation of YAP-1, supporting that the subcellular localization of YAP-1 is regulated in a similar way as that of YAP. Heat shock also causes the nuclear accumulation of YAP-1 but after heat shock, YAP-1 translocates to cytoplasm. Knockdowns of DAF-21 (HSP90 ortholog) and HSF-1block the nuclear export of YAP-1 during this recovery. YAP-1 overexpression is beneficial for thermotolerance, whereas YAP-1 hyperactivity induced by wts-1 and ftt-2 knockdowns is deleterious on thermal response and yap-1 deficiency promotes health aging. In short, YAP-1 partially shares basal characters with mammalian YAP and plays a role in thermal stress response and healthy aging. - Highlights: ► We named Caenorhabditis elegans F13E6.4 gene yap-1 as a putative YAP homolog. ► The localization of YAP-1 is regulated by WTS-1 and FTT-2. ► YAP-1 is involved in healthy aging and thermosensitivity.

  13. Binding Mode Prediction of 5-Hydroxytryptamine 2C Receptor Ligands by Homology Modeling and Molecular Docking Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Asif; Nagarajan, Shanthi; Doddareddy, Munikumar Reddy; Cho, Yong Seo; Pae, Ae Nim [Korea Institute of Science and Technology, Seoul (Korea, Republic of)

    2011-06-15

    Serotonin or 5-hydroxytryptamine subtype 2C (5-HT{sub 2C}) receptor belongs to class A amine subfamily of Gprotein- coupled receptor (GPCR) super family and its ligands has therapeutic promise as anti-depressant and -obesity agents. So far, bovine rhodopsin from class A opsin subfamily was the mostly used X-ray crystal template to model this receptor. Here, we explained homology model using beta 2 adrenergic receptor (β2AR), the model was energetically minimized and validated by flexible ligand docking with known agonists and antagonists. In the active site Asp134, Ser138 of transmembrane 3 (TM3), Arg195 of extracellular loop 2 (ECL2) and Tyr358 of TM7 were found as important residues to interact with agonists. In addition to these, V208 of ECL2 and N351 of TM7 was found to interact with antagonists. Several conserved residues including Trp324, Phe327 and Phe328 were also found to contribute hydrophobic interaction. The predicted ligand binding mode is in good agreement with published mutagenesis and homology model data. This new template derived homology model can be useful for further virtual screening based lead identification.

  14. Discovery of novel inhibitors of Mycobacterium tuberculosis MurG: homology modelling, structure based pharmacophore, molecular docking, and molecular dynamics simulations.

    Science.gov (United States)

    Saxena, Shalini; Abdullah, Maaged; Sriram, Dharmarajan; Guruprasad, Lalitha

    2017-10-17

    MurG (Rv2153c) is a key player in the biosynthesis of the peptidoglycan layer in Mycobacterium tuberculosis (Mtb). This work is an attempt to highlight the structural and functional relationship of Mtb MurG, the three-dimensional (3D) structure of protein was constructed by homology modelling using Discovery Studio 3.5 software. The quality and consistency of generated model was assessed by PROCHECK, ProSA and ERRAT. Later, the model was optimized by molecular dynamics (MD) simulations and the optimized model complex with substrate Uridine-diphosphate-N-acetylglucosamine (UD1) facilitated us to employ structure-based virtual screening approach to obtain new hits from Asinex database using energy-optimized pharmacophore modelling (e-pharmacophore). The pharmacophore model was validated using enrichment calculations, and finally, validated model was employed for high-throughput virtual screening and molecular docking to identify novel Mtb MurG inhibitors. This study led to the identification of 10 potential compounds with good fitness, docking score, which make important interactions with the protein active site. The 25 ns MD simulations of three potential lead compounds with protein confirmed that the structure was stable and make several non-bonding interactions with amino acids, such as Leu290, Met310 and Asn167. Hence, we concluded that the identified compounds may act as new leads for the design of Mtb MurG inhibitors.

  15. C. elegans ring finger protein RNF-113 is involved in interstrand DNA crosslink repair and interacts with a RAD51C homolog.

    Directory of Open Access Journals (Sweden)

    Hyojin Lee

    Full Text Available The Fanconi anemia (FA pathway recognizes interstrand DNA crosslinks (ICLs and contributes to their conversion into double-strand DNA breaks, which can be repaired by homologous recombination. Seven orthologs of the 15 proteins associated with Fanconi anemia are functionally conserved in the model organism C. elegans. Here we report that RNF-113, a ubiquitin ligase, is required for RAD-51 focus formation after inducing ICLs in C. elegans. However, the formation of foci of RPA-1 or FCD-2/FANCD2 in the FA pathway was not affected by depletion of RNF-113. Nevertheless, the RPA-1 foci formed did not disappear with time in the depleted worms, implying serious defects in ICL repair. As a result, RNF-113 depletion increased embryonic lethality after ICL treatment in wild-type worms, but it did not increase the ICL-induced lethality of rfs-1/rad51C mutants. In addition, the persistence of RPA-1 foci was suppressed in doubly-deficient rnf-113;rfs-1 worms, suggesting that there is an epistatic interaction between the two genes. These results lead us to suggest that RNF-113 and RFS-1 interact to promote the displacement of RPA-1 by RAD-51 on single-stranded DNA derived from ICLs.

  16. QSAR models for prediction of chromatographic behavior of homologous Fab variants.

    Science.gov (United States)

    Robinson, Julie R; Karkov, Hanne S; Woo, James A; Krogh, Berit O; Cramer, Steven M

    2017-06-01

    While quantitative structure activity relationship (QSAR) models have been employed successfully for the prediction of small model protein chromatographic behavior, there have been few reports to date on the use of this methodology for larger, more complex proteins. Recently our group generated focused libraries of antibody Fab fragment variants with different combinations of surface hydrophobicities and electrostatic potentials, and demonstrated that the unique selectivities of multimodal resins can be exploited to separate these Fab variants. In this work, results from linear salt gradient experiments with these Fabs were employed to develop QSAR models for six chromatographic systems, including multimodal (Capto MMC, Nuvia cPrime, and two novel ligand prototypes), hydrophobic interaction chromatography (HIC; Capto Phenyl), and cation exchange (CEX; CM Sepharose FF) resins. The models utilized newly developed "local descriptors" to quantify changes around point mutations in the Fab libraries as well as novel cluster descriptors recently introduced by our group. Subsequent rounds of feature selection and linearized machine learning algorithms were used to generate robust, well-validated models with high training set correlations (R 2  > 0.70) that were well suited for predicting elution salt concentrations in the various systems. The developed models then were used to predict the retention of a deamidated Fab and isotype variants, with varying success. The results represent the first successful utilization of QSAR for the prediction of chromatographic behavior of complex proteins such as Fab fragments in multimodal chromatographic systems. The framework presented here can be employed to facilitate process development for the purification of biological products from product-related impurities by in silico screening of resin alternatives. Biotechnol. Bioeng. 2017;114: 1231-1240. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins

    DEFF Research Database (Denmark)

    Bruun, A W; Svendsen, I; Sørensen, S O

    1998-01-01

    signals for transport into the endoplasmic reticulum. Surprisingly, Ic is encoded by TFS1, which has previously been isolated as a high-copy suppressor of cdc25-1. CDC25 encodes the putative GTP exchange factor for Ras1p/Ras2p in yeast. In an attempt to rationalize this finding, we looked...... degree of specificity, showing a 200-fold higher Ki toward a carboxypeptidase from Candida albicans which is highly homologous to carboxypeptidase Y. The TFS1 gene product shows extensive similarity to a class of proteins termed "21-23-kDa lipid binding proteins", members of which are found in several...

  18. Analysis of the expression level and methylation of tumor protein p53, phosphatase and tensin homolog and mutS homolog 2 in N-methyl-N-nitrosourea-induced thymic lymphoma in C57BL/6 mice.

    Science.gov (United States)

    Huo, Xueyun; Li, Zhenkun; Zhang, Shuangyue; Li, Changlong; Guo, Meng; Lu, Jing; Lv, Jianyi; Du, Xiaoyan; Chen, Zhenwen

    2017-10-01

    Tumorigenesis is often caused by somatic mutation or epigenetic changes in genes that regulate aspects of cell death, proliferation and survival. Although the functions of multiple tumor suppressor genes have been well studied in isolation, how these genes cooperate during the progression of a single tumor remains unclear in numerous cases. The present study used N-methyl-N-nitrosourea (MNU), one of the most potent mutagenic nitrosourea compounds, to induce thymic lymphoma in C57BL/6J mice. Subsequently, the protein expression levels of phosphatase and tensin homolog (PTEN), transformation protein 53 and mutS homolog 2 (MSH2) were evaluated concomitantly in the thymus, liver, kidney and spleen of MNU-treated mice by western blotting. To determine whether changes in expression level were due to aberrant epigenetic regulation, the present study further examined the methylation status of each gene by MassARRAY analysis. During the tumorigenesis process of an MNU-induced single thymic lymphoma, the expression level of PTEN was revealed to be reduced in thymic lymphoma samples but not in normal or non-tumor thymus tissue samples. Furthermore, a marked reduction of P53 expression levels were demonstrated in thymic lymphomas and spleens with a metastatic tumor. Conversely, MSH2 upregulation was identified only in liver, kidney, and spleen samples that were infiltrated by thymic lymphoma cells. Furthermore, the present study revealed that a number of 5'-C-phosphate-G-3' sites located in the promoter of aberrantly expressed genes had significantly altered methylation statuses. These results improve the understanding of the course of mutagen-induced cancer, and highlight that epigenetic regulation may serve an important function in cancer.

  19. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

    International Nuclear Information System (INIS)

    Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A.; Johnson, D.I.; Evans, T.

    1990-01-01

    The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G p (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G p protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein

  20. Homology modelling and docking studies on Neuraminidase enzyme as a natural product target for combating influenza

    Directory of Open Access Journals (Sweden)

    Nisha Singh

    2017-10-01

    Full Text Available Influenza remains to be dreadful with yearly epidemics and sudden pandemic outbreaks causing significant mortality, even in nations with the most advanced health care systems. Thus, there has been a long-standing interest to develop effective and safe antiviral agents to treat infected individuals. Attempt to identify suitable molecular targets as antiviral compounds have focused recently on the influenza virus neuraminidase (NA, a key enzyme in viral replication [1]. In this research, virtual screening was done on a total of 600 natural compounds from 22 ethno medicinal Indian herbs for activity against neuraminidase enzyme exploiting representative protein conformations selected from molecular dynamics simulations. Neuraminidase enzyme sequences from different existing strains available on National Center of Biotechnology Information [2] (NCBI protein database were aligned using Clustal W [3] and CLC workbench 10 [4] to find the conserved residues. Neuraminidase protein sequence from H1N1 strain available on NCBI was used to structure 3D target model predicted against dataset from Protein data bank using modeller [5]. The target model was validated on different parameter at SAVES Server [6]. Using this target model a pharmacophore model was developed using ligand based strategy exploiting the three known inhibitors. The docking parameters were validated by redocking Zanamivir to its co-complex 2009 H1N1 NA crystal structure (PDB ID: 3TI5 generating best pose with a RMSD value of 0.7543 A°. This model was then used for in silico analysis of a library of natural compounds from 22 ethno medicinal Indian herbs known to have antiviral activity taken downloaded from PubChem database and selected on the basis of drug likeliness. All the compounds were docked in the binding pocket of neuraminidase. Top compounds having binding affinity better than or comparable to the control drug Zanamivir were selected and analyzed for their ADME and toxicity

  1. Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral.

    Science.gov (United States)

    Smith-Keune, C; Dove, S

    2008-01-01

    Recent incidences of mass coral bleaching indicate that major reef building corals are increasingly suffering thermal stress associated with climate-related temperature increases. The development of pulse amplitude modulated (PAM) fluorometry has enabled rapid detection of the onset of thermal stress within coral algal symbionts, but sensitive biomarkers of thermal stress specific to the host coral have been slower to emerge. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to produce fingerprints of gene expression for the reef-building coral Acropora millepora exposed to 33 degrees C. Changes in the expression of 23 out of 399 putative genes occurred within 144 h. Down-regulation of one host-specific gene (AmA1a) occurred within just 6 h. Full-length sequencing revealed the product of this gene to be an all-protein chromatophore (green fluorescent protein [GFP]-homolog). RT-PCR revealed consistent down-regulation of this GFP-homolog for three replicate colonies within 6 h at both 32 degrees C and 33 degrees C but not at lower temperatures. Down-regulation of this host gene preceded significant decreases in the photosynthetic activity of photosystem II (dark-adapted F (v)/F (m)) of algal symbionts as measured by PAM fluorometry. Gene expression of host-specific genes such as GFP-homologs may therefore prove to be highly sensitive indicators for the onset of thermal stress within host coral cells.

  2. Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Ngo, Hien-Ping; Hickson, Ian D

    2007-01-01

    CSM2, PSY3, SHU1, and SHU2 (collectively referred to as the SHU genes) were identified in Saccharomyces cerevisiae as four genes in the same epistasis group that suppress various sgs1 and top3 mutant phenotypes when mutated. Although the SHU genes have been implicated in homologous recombination ...

  3. Induction of the unfolded protein response by cigarette smoke is primarily an activating transcription factor 4-C/EBP homologous protein mediated process

    Directory of Open Access Journals (Sweden)

    Geraghty P

    2011-06-01

    Full Text Available Patrick Geraghty, Alison Wallace, Jeanine M D'ArmientoDepartment of Medicine, Divisions of Molecular and Pulmonary Medicine, Columbia University College of Physicians and Surgeons, New York, NY, USAPurpose: Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease (COPD. Recent studies propose a link between endoplasmic reticulum (ER stress and emphysema, demonstrated by increased ER stress markers under smoking conditions. Here, we investigate whether cigarette smoke-induced ER stress is cell specific and correlates with acute and chronic cigarette smoke exposure.Methods: Gene and protein expression changes in human primary lung cell cultures following cigarette smoke extract (CSE exposure were monitored by qPCR and Western blot analysis. Mice and guinea pigs were exposed to cigarette smoke and ER stress markers examined in whole lung homogenates. Inflammatory cells from the bronchoalveolar lavage fluid of 10 days smoke exposed mice were also examined.Results: Cigarette smoke induced a trend increase in the ER stress response through an activating transcription factor 4 (ATF4 mediated induction of C/EBP homologous protein (CHOP in primary small airway epithelial cells. Bronchial epithelial cells and macrophages responded similarly to CSE. Wild-type mice and guinea pigs exposed to acute levels of cigarette smoke exhibited increased levels of CHOP but not at significant levels. However, after long-term chronic cigarette smoke exposure, CHOP expression was reduced. Interestingly, inflammatory cells from smoke exposed mice had a significant increase in CHOP/ATF4 expression.Conclusion: A trend increase in CHOP levels appear in multiple human lung cell types following acute cigarette smoke exposure in vitro. In vivo, inflammatory cells, predominately macrophages, demonstrate significant cigarette smoke-induced ER stress. Early induction of CHOP in cigarette smoke may play a pivotal role in early

  4. Mycobacterium tuberculosis and Mycobacterium marinum non-homologous end-joining proteins can function together to join DNA ends in Escherichia coli.

    Science.gov (United States)

    Wright, Douglas G; Castore, Reneau; Shi, Runhua; Mallick, Amrita; Ennis, Don G; Harrison, Lynn

    2017-03-01

    Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). This pathway protects against DNA damage when bacteria are in stationary phase. Mycobacterium marinum is a member of this mycobacterium family and like M. tuberculosis is pathogenic. M. marinum lives in water, forms biofilms and infects fish and frogs. M. marinum is a biosafety level 2 (BSL2) organism as it can infect humans, although infections are limited to the skin. M. marinum is accepted as a model to study mycobacterial pathogenesis, as M. marinum and M. tuberculosis are genetically closely related and have similar mechanisms of survival and persistence inside macrophage. The aim of this study was to determine whether M. marinum could be used as a model to understand M. tuberculosis NHEJ repair. We identified and cloned the M. marinum genes encoding NHEJ proteins and generated E. coli strains that express the M. marinum Ku (Mm-Ku) and ligase D (Mm-Lig) individually or together (LHmKumLig strain) from expression vectors integrated at phage attachment sites in the genome. We demonstrated that Mm-Ku and Mm-Lig are both required to re-circularize Cla I-linearized plasmid DNA in E. coli. We compared repair of strain LHmKumLig with that of an E. coli strain (BWKuLig#2) expressing the M. tuberculosis Ku (Mt-Ku) and ligase D (Mt-Lig), and found that LHmKumLig performed 3.5 times more repair and repair was more accurate than BWKuLig#2. By expressing the Mm-Ku with the Mt-Lig, or the Mt-Ku with the Mm-Lig in E. coli, we have shown that the NHEJ proteins from M. marinum and M. tuberculosis can function together to join DNA DSBs. NHEJ repair is therefore conserved between the two species. Consequently, M. marinum is a good model to study NHEJ repair during mycobacterial pathogenesis. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen

  5. A role for the malignant brain tumour (MBT domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    Directory of Open Access Journals (Sweden)

    Nicholas M Johnson

    Full Text Available Malignant brain tumour (MBT domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR for the repair of DNA double-strand breaks (DSBs. lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  6. The PMDB Protein Model Database

    Science.gov (United States)

    Castrignanò, Tiziana; De Meo, Paolo D'Onorio; Cozzetto, Domenico; Talamo, Ivano Giuseppe; Tramontano, Anna

    2006-01-01

    The Protein Model Database (PMDB) is a public resource aimed at storing manually built 3D models of proteins. The database is designed to provide access to models published in the scientific literature, together with validating experimental data. It is a relational database and it currently contains >74 000 models for ∼240 proteins. The system is accessible at and allows predictors to submit models along with related supporting evidence and users to download them through a simple and intuitive interface. Users can navigate in the database and retrieve models referring to the same target protein or to different regions of the same protein. Each model is assigned a unique identifier that allows interested users to directly access the data. PMID:16381873

  7. Construction of Lactococcus lactis expressing secreted and anchored Eimeria tenella 3-1E protein and comparison of protective immunity against homologous challenge.

    Science.gov (United States)

    Ma, Chunli; Zhang, Lili; Gao, Mingyang; Ma, Dexing

    2017-07-01

    Two novel plasmids pTX8048-SP-Δ3-1E and pTX8048-SP-NAΔ3-1E-CWA were constructed. The plasmids were respectively electrotransformed into L. lactis NZ9000 to generate strain of L. lactis/pTX8048-SP-Δ3-1E in which 3-1E protein was expressed in secretion, and L. lactis/pTX8048-SP-NAΔ3-1E-CWA on which 3-1E protein was covalently anchored to the surface of bacteria cells. The expression of target proteins were examined by Western blot. The live lactococci expressing secreted 3-1E protein, anchored 3-1E protein, and cytoplasmic 3-1E protein was administered orally to chickens respectively, and the protective immunity and efficacy were compared by animal experiment. The results showed oral immunization to chickens with recombinant lactococci expressing anchored 3-1E protein elicited high 3-1E-specific serum IgG, increased high proportion of CD4 + and CD8α + cells in spleen, alleviated average lesion score in cecum, decreased the oocyst output per chicken compared to lactococci expressing cytoplasmic or secreted 3-1E protein. Taken together, these findings indicated the surface anchored Eimeria protein displayed by L. lacits can induce protective immunity and partial protection against homologous infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Combined HQSAR, topomer CoMFA, homology modeling and docking studies on triazole derivatives as SGLT2 inhibitors.

    Science.gov (United States)

    Yu, Shuling; Yuan, Jintao; Zhang, Yi; Gao, Shufang; Gan, Ying; Han, Meng; Chen, Yuewen; Zhou, Qiaoqiao; Shi, Jiahua

    2017-06-01

    Sodium-glucose cotransporter 2 (SGLT2) is a promising target for diabetes therapy. We aimed to develop computational approaches to identify structural features for more potential SGLT2 inhibitors. In this work, 46 triazole derivatives as SGLT2 inhibitors were studied using a combination of several approaches, including hologram quantitative structure-activity relationships (HQSAR), topomer comparative molecular field analysis (CoMFA), homology modeling, and molecular docking. HQSAR and topomer CoMFA were used to construct models. Molecular docking was conducted to investigate the interaction of triazole derivatives and homology modeling of SGLT2, as well as to validate the results of the HQSAR and topomer CoMFA models. The most effective HQSAR and topomer CoMFA models exhibited noncross-validated correlation coefficients of 0.928 and 0.891 for the training set, respectively. External predictions were made successfully on a test set and then compared with previously reported models. The graphical results of HQSAR and topomer CoMFA were proven to be consistent with the binding mode of the inhibitors and SGLT2 from molecular docking. The models and docking provided important insights into the design of potent inhibitors for SGLT2.

  9. A homologous mapping method for three-dimensional reconstruction of protein networks reveals disease-associated mutations.

    Science.gov (United States)

    Huang, Sing-Han; Lo, Yu-Shu; Luo, Yong-Chun; Tseng, Yu-Yao; Yang, Jinn-Moon

    2018-03-19

    One of the crucial steps toward understanding the associations among molecular interactions, pathways, and diseases in a cell is to investigate detailed atomic protein-protein interactions (PPIs) in the structural interactome. Despite the availability of large-scale methods for analyzing PPI networks, these methods often focused on PPI networks using genome-scale data and/or known experimental PPIs. However, these methods are unable to provide structurally resolved interaction residues and their conservations in PPI networks. Here, we reconstructed a human three-dimensional (3D) structural PPI network (hDiSNet) with the detailed atomic binding models and disease-associated mutations by enhancing our PPI families and 3D-domain interologs from 60,618 structural complexes and complete genome database with 6,352,363 protein sequences across 2274 species. hDiSNet is a scale-free network (γ = 2.05), which consists of 5177 proteins and 19,239 PPIs with 5843 mutations. These 19,239 structurally resolved PPIs not only expanded the number of PPIs compared to present structural PPI network, but also achieved higher agreement with gene ontology similarities and higher co-expression correlation than the ones of 181,868 experimental PPIs recorded in public databases. Among 5843 mutations, 1653 and 790 mutations involved in interacting domains and contacting residues, respectively, are highly related to diseases. Our hDiSNet can provide detailed atomic interactions of human disease and their associated proteins with mutations. Our results show that the disease-related mutations are often located at the contacting residues forming the hydrogen bonds or conserved in the PPI family. In addition, hDiSNet provides the insights of the FGFR (EGFR)-MAPK pathway for interpreting the mechanisms of breast cancer and ErbB signaling pathway in brain cancer. Our results demonstrate that hDiSNet can explore structural-based interactions insights for understanding the mechanisms of disease

  10. Homology modeling of Leishmania donovani enolase and its molecular interaction with novel inhibitors

    Directory of Open Access Journals (Sweden)

    Jay Prakash Mahato

    2017-01-01

    Full Text Available Introduction: The treatment of Indian tropical disease such as kala-azar is likely to be troublesome to the clinicians as AmpB- and miltefosine-resistant Leishmania donovani has been reported. The rationale behind designed a novel inhibitors of model of L. donovani enolase and performing a binding study with its inhibitors to gain details of the interaction between protein residues and ligand molecules. Methods and Materials: The L. donovani enolase model consists of two typical domains. The N-terminal one contains three-stranded antiparallel β-sheets, followed by six α-helices. The C-terminal domain composes of eleven-stranded mixed α/β-barrel with connectivity. The first α-helix within the C-terminal domain, H7, and the second β-strand, S7, of the barrel domain was arranged in an antiparallel fashion compared to all other α-helices and β-strands. The root-mean-square deviation between predicted model and template is 0.4 Å. The overall conformation of L. donovani enolase model is similar to those of Trypanosoma cruzi enolase and Streptococcus pneumoniae enolase crystal structures. Result: The key amino acid residues within the docking complex model involved in the interaction between model enolase structure and ligand molecule are Lys70, Asn165, Ala168, Asp17, and Asn213. Conclusion: Our theoretical prediction may lead to the establishment of prophylactic and therapeutic approaches for the treatment of kala-azar. This biomedical informatics analysis will help us to combat future kala-azar.

  11. Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

    Science.gov (United States)

    Ciarlet, Max; Estes, Mary K.; Barone, Christopher; Ramig, Robert F.; Conner, Margaret E.

    1998-01-01

    The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 103 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction

  12. The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

    Science.gov (United States)

    Fujinami, Shun; Ito, Masahiro

    2018-01-01

    It is well known that the Na + cycle and the cell wall are essential for alkaline adaptation of Na + -dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na + concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na + concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

  13. Comments on the Updated Tetrapartite Pallium Model in the Mouse and Chick, Featuring a Homologous Claustro-Insular Complex.

    Science.gov (United States)

    Puelles, Luis

    2017-01-01

    This essay reviews step by step the conceptual changes of the updated tetrapartite pallium model from its tripartite and early tetrapartite antecedents. The crucial observations in mouse material are explained first in the context of assumptions, tentative interpretations, and literature data. Errors and the solutions offered to resolve them are made explicit. Next, attention is centered on the lateral pallium sector of the updated model, whose definition is novel in incorporating a claustro-insular complex distinct from both olfactory centers (ventral pallium) and the isocortex (dorsal pallium). The general validity of the model is postulated at least for tetrapods. Genoarchitectonic studies performed to check the presence of a claustro-insular field homolog in the avian brain are reviewed next. These studies have indeed revealed the existence of such a complex in the avian mesopallium (though stratified outside-in rather than inside-out as in mammals), and there are indications that the same pattern may be found in reptiles as well. Peculiar pallio-pallial tangential migratory phenomena are apparently shared as well between mice and chicks. The issue of whether the avian mesopallium has connections that are similar to the known connections of the mammalian claustro-insular complex is considered next. Accrued data are consistent with similar connections for the avian insula homolog, but they are judged to be insufficient to reach definitive conclusions about the avian claustrum. An aside discusses that conserved connections are not a necessary feature of field-homologous neural centers. Finally, the present scenario on the evolution of the pallium of sauropsids and mammals is briefly visited, as highlighted by the updated tetrapartite model and present results. © 2017 S. Karger AG, Basel.

  14. Validation of a homology model of Mycobacterium tuberculosis DXS: rationalization of observed activities of thiamine derivatives as potent inhibitors of two orthologues of DXS.

    Science.gov (United States)

    Masini, T; Lacy, B; Monjas, L; Hawksley, D; de Voogd, A R; Illarionov, B; Iqbal, A; Leeper, F J; Fischer, M; Kontoyianni, M; Hirsch, A K H

    2015-12-14

    The enzyme DXS catalyzes the first, rate-limiting step of the 2-C-methyl-d-erythritol-4-phosphate (MEP, 1) pathway using thiamine diphosphate (ThDP) as cofactor; the DXS-catalyzed reaction constitutes also the first step in vitamin B1 and B6 metabolism in bacteria. DXS is the least studied among the enzymes of this pathway in terms of crystallographic information, with only one complete crystal structure deposited in the Protein Data Bank (Deinococcus radiodurans DXS, PDB: ). We synthesized a series of thiamine and ThDP derivatives and tested them for their biochemical activity against two DXS orthologues, namely D. radiodurans DXS and Mycobacterium tuberculosis DXS. These experimental results, combined with advanced docking studies, led to the development and validation of a homology model of M. tuberculosis DXS, which, in turn, will guide medicinal chemists in rationally designing potential inhibitors for M. tuberculosis DXS.

  15. Inter-homolog crossing-over and synapsis in Arabidopsis meiosis are dependent on the chromosome axis protein AtASY3.

    Directory of Open Access Journals (Sweden)

    Maheen Ferdous

    2012-02-01

    Full Text Available In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs. Although the frequency of DNA double-strand breaks (DSBs appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR, we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.

  16. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

    DEFF Research Database (Denmark)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia

    2016-01-01

    to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during...... recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits...

  17. echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

    Directory of Open Access Journals (Sweden)

    Gorski Sharon M

    2007-07-01

    Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

  18. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

    2006-01-01

    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly ...

  19. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

  20. HHR23A, a human homolog of Saccharomyces cerevisiae Rad23, regulates xeroderma pigmentosum C protein and is required for nucleotide excision repair

    International Nuclear Information System (INIS)

    Hsieh, Hui-Chuan; Hsieh, Yi-Hsuan; Huang, Yu-Hsin; Shen, Fan-Ching; Tsai, Han-Ni; Tsai, Jui-He; Lai, Yu-Ting; Wang, Yu-Ting; Chuang, Woei-Jer; Huang, Wenya

    2005-01-01

    HHR23A and hHR23B are the human homologs of Saccharomyces cerevisiae Rad23. hHR23B is associated with the nucleotide excision repair (NER) factor xeroderma pigmentosum C (XPC) protein and is required for global genome repair. The function of hHR23A is not yet clear. In this study, the potential function of the hHR23A protein was investigated using RNA interference techniques. The hHR23A knock-down (KD) construct diminished the RNA level of hHR23A protein by approximately 60%, and it did not interfere with expression of the hHR23B gene. Based on Southwestern immunoblot and host-cell reactivation assays, hHR23A KD cells were found to be deficient in DNA repair activity against the DNA damage caused by UVC irradiation. In these hHR23A KD cells, the XPC gene was not normally induced by UVC irradiation, indicating that the hHR23A protein is involved in NER through regulation of the DNA damage recognition protein XPC. Co-immunoprecipitation experiments revealed that hHR23A was associated with a small portion of hHR23B and the majority of p53 protein, indicating that hHR23A regulates the function of XPC by its association with the NER activator p53

  1. Crystallization and preliminary crystallographic analysis of the human calcineurin homologous protein CHP2 bound to the cytoplasmic region of the Na+/H+ exchanger NHE1

    International Nuclear Information System (INIS)

    Ben Ammar, Youssef; Takeda, Soichi; Sugawara, Mitsuaki; Miyano, Masashi; Mori, Hidezo; Wakabayashi, Shigeo

    2005-01-01

    Crystallization of the human CHP2–NHE1 binding domain complex. Calcineurin homologous protein (CHP) is a Ca 2+ -binding protein that directly interacts with and regulates the activity of all plasma-membrane Na + /H + -exchanger (NHE) family members. In contrast to the ubiquitous isoform CHP1, CHP2 is highly expressed in cancer cells. To understand the regulatory mechanism of NHE1 by CHP2, the complex CHP2–NHE1 (amino acids 503–545) has been crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as precipitant. The crystals diffract to 2.7 Å and belong to a tetragonal space group, with unit-cell parameters a = b = 49.96, c = 103.20 Å

  2. Expression of venom gene homologs in diverse python tissues suggests a new model for the evolution of snake venom.

    Science.gov (United States)

    Reyes-Velasco, Jacobo; Card, Daren C; Andrew, Audra L; Shaney, Kyle J; Adams, Richard H; Schield, Drew R; Casewell, Nicholas R; Mackessy, Stephen P; Castoe, Todd A

    2015-01-01

    Snake venom gene evolution has been studied intensively over the past several decades, yet most previous studies have lacked the context of complete snake genomes and the full context of gene expression across diverse snake tissues. We took a novel approach to studying snake venom evolution by leveraging the complete genome of the Burmese python, including information from tissue-specific patterns of gene expression. We identified the orthologs of snake venom genes in the python genome, and conducted detailed analysis of gene expression of these venom homologs to identify patterns that differ between snake venom gene families and all other genes. We found that venom gene homologs in the python are expressed in many different tissues outside of oral glands, which illustrates the pitfalls of using transcriptomic data alone to define "venom toxins." We hypothesize that the python may represent an ancestral state prior to major venom development, which is supported by our finding that the expansion of venom gene families is largely restricted to highly venomous caenophidian snakes. Therefore, the python provides insight into biases in which genes were recruited for snake venom systems. Python venom homologs are generally expressed at lower levels, have higher variance among tissues, and are expressed in fewer organs compared with all other python genes. We propose a model for the evolution of snake venoms in which venom genes are recruited preferentially from genes with particular expression profile characteristics, which facilitate a nearly neutral transition toward specialized venom system expression. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

    OpenAIRE

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high Pr...

  4. AHM1, a Novel Type of Nuclear Matrix–Localized, MAR Binding Protein with a Single AT Hook and a J Domain–Homologous Region

    Science.gov (United States)

    Morisawa, Gaku; Han-yama, Atsushi; Moda, Ichiro; Tamai, Atsushi; Iwabuchi, Masaki; Meshi, Tetsuo

    2000-01-01

    Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook–containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain–homologous region and a Zn finger–like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger–like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. PMID:11041885

  5. Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

    Science.gov (United States)

    Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E.; Zickler, Denise; Espagne, Eric

    2014-01-01

    Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly. PMID:25210014

  6. FeatureMap3D - a tool to map protein features and sequence conservation onto homologous structures in the PDB

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Rapacki, Krzysztof; Stærfeldt, Hans Henrik

    2006-01-01

    FeatureMap3D is a web-based tool that maps protein features onto 3D structures. The user provides sequences annotated with any feature of interest, such as post-translational modifications, protease cleavage sites or exonic structure and FeatureMap3D will then search the Protein Data Bank (PDB) f...

  7. Anomalous diffusion in neutral evolution of model proteins

    Science.gov (United States)

    Nelson, Erik D.; Grishin, Nick V.

    2015-06-01

    Protein evolution is frequently explored using minimalist polymer models, however, little attention has been given to the problem of structural drift, or diffusion. Here, we study neutral evolution of small protein motifs using an off-lattice heteropolymer model in which individual monomers interact as low-resolution amino acids. In contrast to most earlier models, both the length and folded structure of the polymers are permitted to change. To describe structural change, we compute the mean-square distance (MSD) between monomers in homologous folds separated by n neutral mutations. We find that structural change is episodic, and, averaged over lineages (for example, those extending from a single sequence), exhibits a power-law dependence on n . We show that this exponent depends on the alignment method used, and we analyze the distribution of waiting times between neutral mutations. The latter are more disperse than for models required to maintain a specific fold, but exhibit a similar power-law tail.

  8. Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis

    Directory of Open Access Journals (Sweden)

    Budzanivska I. G.

    2014-03-01

    Full Text Available Aim. Identification of the widespread Ukrainian isolate(s of PVY (Potato virus Y in different potato cultivars and subsequent phylogenetic analysis of detected PVY isolates based on NA and AA sequences of coat protein. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. PVY has been identified serologically in potato cultivars of Ukrainian selection. In this work we have optimized a method for total RNA extraction from potato samples and offered a sensitive and specific PCR-based test system of own design for diagnostics of the Ukrainian PVY isolates. Part of the CP gene of the Ukrainian PVY isolate has been sequenced and analyzed phylogenetically. It is demonstrated that the Ukrainian isolate of Potato virus Y (CP gene has a higher percentage of homology with the recombinant isolates (strains of this pathogen (approx. 98.8– 99.8 % of homology for both nucleotide and translated amino acid sequences of the CP gene. The Ukrainian isolate of PVY is positioned in the separate cluster together with the isolates found in Syria, Japan and Iran; these isolates possibly have common origin. The Ukrainian PVY isolate is confirmed to be recombinant. Conclusions. This work underlines the need and provides the means for accurate monitoring of Potato virus Y in the agroecosystems of Ukraine. Most importantly, the phylogenetic analysis demonstrated the recombinant nature of this PVY isolate which has been attributed to the strain group O, subclade N:O.

  9. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair.

    Science.gov (United States)

    Kato-Inui, Tomoko; Takahashi, Gou; Hsu, Szuyin; Miyaoka, Yuichiro

    2018-05-18

    Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established. We systematically analyzed the HDR and NHEJ products after genome editing using various modified guide RNAs (gRNAs) and Cas9 variants with an enhanced conformational checkpoint to improve the fidelity at endogenous gene loci in HEK293T cells and HeLa cells. We found that these modified gRNAs and Cas9 variants were able to enhance HDR in both single-nucleotide substitutions and a multi-kb DNA fragment insertion. Our results suggest that the original CRISPR/Cas9 system from the bacterial immune system is not necessarily the best option for the induction of HDR in genome editing and indicate that the modulation of the kinetics of conformational checkpoints of Cas9 can optimize the HDR/NHEJ ratio.

  10. The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells.

    Science.gov (United States)

    Agizzio, Ana Paula; Da Cunha, Maura; Carvalho, André O; Oliveira, Marco Antônio; Ribeiro, Suzanna F F; Gomes, Valdirene M

    2006-10-01

    Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles. Copyright © 2006 Elsevier Ireland Ltd. All rights reserved.

  11. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  12. Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins*

    Science.gov (United States)

    Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

    2017-01-01

    Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. PMID:27927989

  13. Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

    Science.gov (United States)

    Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

    2017-01-20

    Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Functional Analysis of Homologous Recombination Repair Proteins HerA and NurA in the Thermophile Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Huang, Qihong

    A number of DNA lesions are generated in each cell every day, among which double-stranded breaks (DSBs) constitute one of the most detrimental types of DNA damage. DSBs lead to genome instability, cell death, or even tumorigenesis in human, if not repaired timely. Two main pathways are known...... in the S/G2 phase of the cell cycle are preferentially repaired by HRR pathway, while NHEJ is the favorate pathway to repair DSBs in the G1 phase. Bacteria encode multiple pathways for DSB repair, including RecBCD, the primary HR pathway, SbcC-SbcD, and one backup system, RecFOR. In eukaryotes, the HRR...... pathway is mediated by Mre11-Rad50, homologs of bacterial SbcD-SbcC. However, numerous proteins and multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts, making many important mechanistic details poorly understood...

  15. Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements

    Directory of Open Access Journals (Sweden)

    van der Oost John

    2009-08-01

    Full Text Available Abstract Background In eukaryotes, RNA interference (RNAi is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. Results We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding and PIWI (active or inactivated nuclease domains, the prokaryotic Argonaute homologs (pAgos fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative nucleases. Given these observations, the apparent extensive

  16. HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

    Science.gov (United States)

    Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Horiguchi, Tomoko; Sun, Xuedong; Deng, Lin; Shoji, Ikuo; Hotta, Hak; Sada, Kiyonao

    2012-01-01

    Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

  17. HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

    Directory of Open Access Journals (Sweden)

    Kenji Nakashima

    Full Text Available Hepatitis C virus (HCV infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV implied that NS5A was tyrosine phosphorylated by pervanadate (PV treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST-fusion proteins of various Src homology 2 (SH2 domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3 domain. Substitution of Arg(176 to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334 was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

  18. Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

    Science.gov (United States)

    Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

    1994-11-04

    Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

  19. The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T) is important for swarming and biofilm formation.

    Science.gov (United States)

    Janesch, Bettina; Koerdt, Andrea; Messner, Paul; Schäffer, Christina

    2013-01-01

    Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB). Paenibacillus alvei CCM 2051(T) is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH) domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA. This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T).

  20. The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T is important for swarming and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Bettina Janesch

    Full Text Available Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB.Paenibacillus alvei CCM 2051(T is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA.This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T.

  1. A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea

    Directory of Open Access Journals (Sweden)

    Venâncio T.M.

    2003-01-01

    Full Text Available Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

  2. Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester.

    Science.gov (United States)

    Chen, Ke-Cai; Zheng, Ming-Min; Pan, Jiang; Li, Chun-Xiu; Xu, Jian-He

    2017-10-01

    The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipA L315S and LipA S271F , were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipA L315S , LipA S271F , and their combination LipA L315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipA S271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipA S271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m . Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.

  3. A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

    Directory of Open Access Journals (Sweden)

    Changrim Lee

    Full Text Available PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs, while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs. In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

  4. The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

    International Nuclear Information System (INIS)

    Senior, Mary M.; Frederick, Anne F.; Black, Stuart; Murgolo, Nicholas J.; Perkins, Louise M.; Wilson, Oswald; Snow, Mark E.; Wang Yusen

    1998-01-01

    A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 A for all backbone atoms and 0.40 A for the backbone atoms of the central β-sheet. The core structure of the SH2 domain contains an antiparallel β-sheet flanked by two parallel α-helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions

  5. Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on diots and monocots

    NARCIS (Netherlands)

    Stergiopoulos, I.; Burg, van den H.A.; Ökmen, B.; Beenen, H.G.; Liere, van S.; Kema, G.H.J.; Wit, de P.J.G.M.

    2010-01-01

    Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce

  6. Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

    Science.gov (United States)

    Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

    2007-01-01

    We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

  7. The Nance-Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology.

    Science.gov (United States)

    Brooks, Simon P; Coccia, Margherita; Tang, Hao R; Kanuga, Naheed; Machesky, Laura M; Bailly, Maryse; Cheetham, Michael E; Hardcastle, Alison J

    2010-06-15

    Nance-Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell-cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development.

  8. The Nance–Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology

    Science.gov (United States)

    Brooks, Simon P.; Coccia, Margherita; Tang, Hao R.; Kanuga, Naheed; Machesky, Laura M.; Bailly, Maryse; Cheetham, Michael E.; Hardcastle, Alison J.

    2010-01-01

    Nance–Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell–cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development. PMID:20332100

  9. Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

    Science.gov (United States)

    Bae, Young-An; Kim, Jeong-Geun; Kong, Yoon

    2016-01-01

    Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. CRTAC1 homolog proteins are conserved from cyanobacteria to man and secreted by the teleost fish pituitary gland.

    Science.gov (United States)

    Redruello, Begoña; Louro, Bruno; Anjos, Liliana; Silva, Nádia; Greenwell, Roger S; Canario, Adelino V M; Power, Deborah M

    2010-05-15

    Cartilage acidic protein 1 (CRTAC1) gene expression is used as a marker for chondrocyte differentiation in stem cell-based tissue engineering. It is also transcribed outside the skeleton where at least two different transcripts are expressed in lung and brain. In the pituitary gland of the teleost fish sea bream Sparus auratus, we have found a transcript with a high degree of sequence identity to CRTAC1 family members but lacking the EGF-like calcium-binding domain encoding sequence of CRTAC1 and designated it as CRTAC2. Database searches revealed many previously unidentified members of the CRTAC1 and CRTAC2 in phylogenetically distant organisms, such as cyanobacteria, bryophyta, lancelets, and diverse representatives of vertebrates. Phylogenetic analyses showed that the genes encoding CRTAC1 and CRTAC2 proteins coexist in teleost fish genomes. Structural prediction analysis identified the N-terminal region of the CRTAC1/CRTAC2 family members as a potential seven-bladed beta-propeller structure, closely related to those of integrin alpha chains and glycosylphosphatidylinositol-specific phospholipase D1 protein families. This relationship is confirmed by phylogenetic analysis with the N-terminal domain of sea bream CRTAC2 as the most divergent sequence. Because teleost fishes are the only phylogenetic group where both CRTAC1 and CRTAC2 genes are present, they occupy a pivotal position in studies of the mechanisms governing the specific expression patterns of each gene/protein subfamily. This will be essential to elucidate their respective biological roles. Copyright 2010 Elsevier B.V. All rights reserved.

  11. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

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    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  12. A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

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    Emanuela Leonardi

    Full Text Available Mutations of human leucine-rich glioma inactivated (LGI1 gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE, a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

  13. Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes[W

    Science.gov (United States)

    Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

    2012-01-01

    Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5′ splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid–dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner. PMID:23192226

  14. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-07-08

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. In silico modelling and validation of differential expressed proteins in lung cancer

    Directory of Open Access Journals (Sweden)

    Bhagavathi S

    2012-05-01

    Full Text Available Objective: The present study aims predict the three dimensional structure of three major proteins responsible for causing Lung cancer. Methods: These are the differentially expressed proteins in lung cancer dataset. Initially, the structural template for these proteins is identified from structural database using homology search and perform homology modelling approach to predict its native 3D structure. Three-dimensional model obtained was validated using Ramachandran plot analysis to find the reliability of the model. Results: Four proteins were differentially expressed and were significant proteins in causing lung cancer. Among the four proteins, Matrixmetallo proteinase (P39900 had a known 3D structure and hence was not considered for modelling. The remaining proteins Polo like kinase I Q58A51, Trophinin B1AKF1, Thrombomodulin P07204 were modelled and validated. Conclusions: The three dimensional structure of proteins provides insights about the functional aspect and regulatory aspect of the protein. Thus, this study will be a breakthrough for further lung cancer related studies.

  16. The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair.

    Directory of Open Access Journals (Sweden)

    Naoki Takahashi

    2010-01-01

    Full Text Available The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

  17. The Vasa Homolog RDE-12 engages target mRNA and multiple argonaute proteins to promote RNAi in C. elegans.

    Science.gov (United States)

    Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C

    2014-04-14

    Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Exploring the role of cellular homologous of the 30K-superfamily of plant virus movement proteins.

    Science.gov (United States)

    Carrasco, José L; Sánchez-Navarro, Jesús A; Elena, Santiago F

    2018-02-21

    Genes orthologous to the 30K-superfamily of movement proteins (MP) from plant viruses have been recently discovered by bioinformatics analyses as integrated elements in the genome of most vascular plants. However, their functional relevance for plants is still unclear. Here, we undertake some preliminary steps into the functional characterization of one of these putative MP genes found in Arabidopsis thaliana. We found that the AtMP gene is expressed at different stages of the plant development, with accumulation being highest in flowers but lowest in mature siliques. We also found down-regulation of the gene may result in a small delay in plant development and in an exacerbation of the negative effect of salinity in germination efficiency. We have also explored whether changes in expression of the endogenous AtMP have any effect on susceptibility to infection with several viruses, and found that the infectivity of tobacco rattle tobravirus was strongly dependent on the expression of the endogenous AtMP. Finally, we have cloned the endogenous MP from four different plant species into an expression vector that allows for specifically assessing their activity as cell-to-cell movement proteins and have shown that though some may still retain the ancestral activity, they do so in a quite inefficient manner, thus suggesting they have acquired a novel function during adaptation to the host genome. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Evolution of the genus Mimivirus based on translation protein homology and its implication in the tree of life.

    Science.gov (United States)

    Marcelino, V M; Espinola, M V P C; Serrano-Solis, V; Farias, S T

    2017-09-27

    The natural history of mimiviruses (i.e., viruses that are members of the Mimivirus genus) is a challenge for modern biology. A new domain of life to include these organisms has been proposed from analysis of gene conservation. We analyzed the evolutionary relationship of proteins involved in the translation system, and our data show that mimiviruses are a sister group of Eukarya. New data about the origins of Eukarya, in which Eukarya appears as a branch derived from the Archaea domain, were discussed, and we suggest that the mimiviruses emerged from the initial population that gave origin to Eukarya and that, in this way are not part of a new domain of life.

  20. Building alternate protein structures using the elastic network model.

    Science.gov (United States)

    Yang, Qingyi; Sharp, Kim A

    2009-02-15

    We describe a method for efficiently generating ensembles of alternate, all-atom protein structures that (a) differ significantly from the starting structure, (b) have good stereochemistry (bonded geometry), and (c) have good steric properties (absence of atomic overlap). The method uses reconstruction from a series of backbone framework structures that are obtained from a modified elastic network model (ENM) by perturbation along low-frequency normal modes. To ensure good quality backbone frameworks, the single force parameter ENM is modified by introducing two more force parameters to characterize the interaction between the consecutive carbon alphas and those within the same secondary structure domain. The relative stiffness of the three parameters is parameterized to reproduce B-factors, while maintaining good bonded geometry. After parameterization, violations of experimental Calpha-Calpha distances and Calpha-Calpha-Calpha pseudo angles along the backbone are reduced to less than 1%. Simultaneously, the average B-factor correlation coefficient improves to R = 0.77. Two applications illustrate the potential of the approach. (1) 102,051 protein backbones spanning a conformational space of 15 A root mean square deviation were generated from 148 nonredundant proteins in the PDB database, and all-atom models with minimal bonded and nonbonded violations were produced from this ensemble of backbone structures using the SCWRL side chain building program. (2) Improved backbone templates for homology modeling. Fifteen query sequences were each modeled on two targets. For each of the 30 target frameworks, dozens of improved templates could be produced In all cases, improved full atom homology models resulted, of which 50% could be identified blind using the D-Fire statistical potential. (c) 2008 Wiley-Liss, Inc.

  1. A Toxoplasma gondii protein with homology to intracellular type Na+/H+ exchangers is important for osmoregulation and invasion

    International Nuclear Information System (INIS)

    Francia, Maria E.; Wicher, Sarah; Pace, Douglas A.; Sullivan, Jack; Moreno, Silvia N.J.; Arrizabalaga, Gustavo

    2011-01-01

    The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na + /H + exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca 2+ concentration [Ca 2+ ] i , and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

  2. Selective Advantage of Recombination in Evolving Protein Populations:. a Lattice Model Study

    Science.gov (United States)

    Williams, Paul D.; Pollock, David D.; Goldstein, Richard A.

    Recent research has attempted to clarify the contributions of several mutational processes, such as substitutions or homologous recombination. Simplistic, tractable protein models, which determine the compact native structure phenotype from the sequence genotype, are well-suited to such studies. In this paper, we use a lattice-protein model to examine the effects of point mutation and homologous recombination on evolving populations of proteins. We find that while the majority of mutation and recombination events are neutral or deleterious, recombination is far more likely to be beneficial. This results in a faster increase in fitness during evolution, although the final fitness level is not significantly changed. This transient advantage provides an evolutionary advantage to subpopulations that undergo recombination, allowing fixation of recombination to occur in the population.

  3. Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

    Science.gov (United States)

    Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg

    2017-10-01

    Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.

  4. Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles.

    Science.gov (United States)

    Teilum, Kaare; Thormann, Thorsten; Caterer, Nigel R; Poulsen, Heidi I; Jensen, Peter H; Knudsen, Jens; Kragelund, Birthe B; Poulsen, Flemming M

    2005-04-01

    Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues. (c) 2005 Wiley-Liss, Inc.

  5. Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

    OpenAIRE

    Kemege, Kyle E.; Hickey, John M.; Barta, Michael L.; Wickstrum, Jason; Balwalli, Namita; Lovell, Scott; Battaile, Kevin P.; Hefty, P. Scott

    2014-01-01

    Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein enco...

  6. A Mesoscopic Model for Protein-Protein Interactions in Solution

    OpenAIRE

    Lund, Mikael; Jönsson, Bo

    2003-01-01

    Protein self-association may be detrimental in biological systems, but can be utilized in a controlled fashion for protein crystallization. It is hence of considerable interest to understand how factors like solution conditions prevent or promote aggregation. Here we present a computational model describing interactions between protein molecules in solution. The calculations are based on a molecular description capturing the detailed structure of the protein molecule using x-ray or nuclear ma...

  7. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Science.gov (United States)

    Zhang, Yang; Devries, Mark E; Skolnick, Jeffrey

    2006-02-01

    G protein-coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha) root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness

  8. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2006-02-01

    Full Text Available G protein-coupled receptors (GPCRs, encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness

  9. Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

    Directory of Open Access Journals (Sweden)

    In Sil Jeong

    Full Text Available Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD PHOSPHATASE-LIKE 1 (CPL1 regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds RNA binding motifs (dsRBMs at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3 as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

  10. Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1

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    Wang Yu

    2011-04-01

    Full Text Available Abstract Background In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV, the causative agent of duck viral enteritis (DVE, the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. Results DEV UL15 consists of two exons with a 3.5 kilobases (kb inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa, whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s in the cytoplasm at 6 h post infection (h p. i. and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s in the cytoplasm in the absence of any other viral protein. Conclusions DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15

  11. Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1.

    Science.gov (United States)

    Zhu, Hongwei; Li, Huixin; Han, Zongxi; Shao, Yuhao; Wang, Yu; Kong, Xiangang

    2011-04-06

    In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein. DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and

  12. Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8

    Directory of Open Access Journals (Sweden)

    Lawal Garba

    2018-03-01

    Full Text Available Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerant Pseudomonas sp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed in Escherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, −6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively

  13. B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro- and anti-apoptotic BCL-2 family proteins.

    Science.gov (United States)

    Renault, Thibaud T; Elkholi, Rana; Bharti, Archana; Chipuk, Jerry E

    2014-09-19

    The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

    NARCIS (Netherlands)

    Siezen, Roland J.; Vos, Willem M. de; Leunissen, Jack A.M.; Dijkstra, Bauke W.

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, >50 subtilases are known, >40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and

  15. HOMOLOGY MODELING AND PROTEIN ENGINEERING STRATEGY OF SUBTILASES, THE FAMILY OF SUBTILISIN-LIKE SERINE PROTEINASES

    NARCIS (Netherlands)

    SIEZEN, RJ; DEVOS, WM; LEUNISSEN, JAM

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, > 50 subtilases are known, > 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase

  16. Solution Structure and Backbone Dynamics of the Pleckstrin Homology Domain of the Human Protein Kinase B (PKB/Akt). Interaction with Inositol Phosphates

    International Nuclear Information System (INIS)

    Auguin, Daniel; Barthe, Philippe; Auge-Senegas, Marie-Therese; Stern, Marc-Henri; Noguchi, Masayuki; Roumestand, Christian

    2004-01-01

    The programmed cell death occurs as part of normal mammalian development. The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 3'-OH kinase (PI3K)/Protein Kinase B (PKB, also named Akt) cascade. Several targets of the PI3K/PKB signaling pathway have been identified that may underlie the ability of this regulatory cascade to promote cell survival. PKB possesses a N-terminal Pleckstrin Homology (PH) domain that binds specifically and with high affinity to PtIns(3,4,5)P 3 and PtIns(3,4)P 2 , the PI3K second messengers. PKB is then recruited to the plasma membrane by virtue of its interaction with 3'-OH phosphatidylinositides and activated. Recent evidence indicates that PKB is active in various types of human cancer; constitutive PKB signaling activation is believed to promote proliferation and increased cell survival, thereby contributing to cancer progression. Thus, it has been shown that induction of PKB activity is augmented by the TCL1/MTCP1 oncoproteins through a physical association requiring the PKB PH domain. Here we present the three-dimensional solution structure of the PH domain of the human protein PKB (isoform β). PKBβ-PH is an electrostatically polarized molecule that adopts the same fold and topology as other PH-domains, consisting of a β-sandwich of seven strands capped on one top by an α-helix. The opposite face presents three variable loops that appear poorly defined in the NMR structure. Measurements of 15 N spin relaxation times and heteronuclear 15 N{ 1 H}NOEs showed that this poor definition is due to intrinsic flexibility, involving complex motions on different time scales. Chemical shift mapping studies correctly defined the binding site of Ins(1,3,4,5)P 4 (the head group of PtIns(3,4,5)P 3 ), as was previously proposed from a

  17. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences

    DEFF Research Database (Denmark)

    Su, J; Yang, L T; Sap, J

    1996-01-01

    domain in Grb2 (, ). We show here that association of Grb2 with RPTPalpha also involves a critical function for the C-terminal SH3 domain of Grb2. Furthermore, Grb2 SH3 binding peptides interfere with RPTPalpha-Grb2 association in vitro, and the RPTPalpha protein can dissociate the Grb2-Sos complex...... in vivo. These observations constitute a novel mode of Grb2 association and suggest a model in which association with a tyrosine-phosphorylated protein restricts the repertoire of SH3 binding proteins with which Grb2 can simultaneously interact. The function of the Tyr798 tyrosine phosphorylation/Grb2...... binding site in RPTPalpha was studied further by expression of wild type or mutant RPTPalpha proteins in PC12 cells. In these cells, wild type RPTPalpha interferes with acidic fibroblast growth factor-induced neurite outgrowth; this effect requires both the catalytic activity and the Grb2 binding Tyr798...

  18. Studies on 16α-Hydroxylation of Steroid Molecules and Regioselective Binding Mode in Homology-Modeled Cytochrome P450-2C11

    Directory of Open Access Journals (Sweden)

    Hamed I. Ali

    2011-01-01

    Full Text Available We investigated the 16α-hydroxylation of steroid molecules and regioselective binding mode in homology-modeled cytochrome P450-2C11 to correlate the biological study with the computational molecular modeling. It revealed that there was a positive relationship between the observed inhibitory potencies and the binding free energies. Docking of steroid molecules into this homology-modeled CYP2C11 indicated that 16α-hydroxylation is favored with steroidal molecules possessing the following components, (1 a bent A-B ring configuration (5β-reduced, (2 C-3 α-hydroxyl group, (3 C-17β-acetyl group, and (4 methyl group at both the C-18 and C-19. These respective steroid components requirements were defined as the inhibitory contribution factor. Overall studies of the male rat CYP2C11 metabolism revealed that the above-mentioned steroid components requirements were essential to induce an effective inhibition of [3H]progesterone 16α-hydroxylation. As far as docking of homology-modeled CYP2C11 against investigated steroids is concerned, they are docked at the active site superimposed with flurbiprofen. It was also found that the distance between heme iron and C16α-H was between 4 to 6 Å and that the related angle was in the range of 180±45∘.

  19. Pure homology of algebraic varieties

    OpenAIRE

    Weber, Andrzej

    2003-01-01

    We show that for a complete complex algebraic variety the pure component of homology coincides with the image of intersection homology. Therefore pure homology is topologically invariant. To obtain slightly more general results we introduce "image homology" for noncomplete varieties.

  20. High prevalence of sensitization to gibberellin-regulated protein (peamaclein) in fruit allergies with negative immunoglobulin E reactivity to Bet v 1 homologs and profilin: Clinical pattern, causative fruits and cofactor effect of gibberellin-regulated protein allergy.

    Science.gov (United States)

    Inomata, Naoko; Miyakawa, Mami; Aihara, Michiko

    2017-07-01

    Gibberellin-regulated protein (GRP) is a new allergen in peach allergy, with an amino acid sequence very well conserved through several botanical species. We investigated the allergenicity of GRP in fruit allergies other than peaches and identified the clinical characteristics of fruit allergy patients with GRP sensitization. One hundred consecutive Japanese patients with fruit allergies were enrolled in the present study. To identify the features of GRP sensitization, we selected patients with negative ImmunoCAP results for Bet v 1 homologs and profilin, which are marker allergens for pollen-food allergy syndrome (PFAS), or lipid transfer protein. These patients underwent specific immunoglobulin E measurements by enzyme-linked immunosorbent assay (ELISA) and skin prick tests (SPT) using purified nPru p 7. Twenty of 100 consecutive patients with fruit allergies had negative ImmunoCAP results for Bet v 1 homologs and profilin. Thirteen (65.0%) of the 20 patients had positive ELISA and/or SPT results using nPru p 7, whereas one of the 20 patients had positive ImmunoCAP results for Pru p 3. In 13 nPru p 7-sensitized patients, the causative foods were peaches (92.3%), apricots (61.5%), oranges (46.2%) and apples (30.8%). Ten patients (76.9%) had multiple causative fruits. Frequent symptoms included facial edema (92.3%) and laryngeal tightness (66.7%). In eight patients (61.5%), exercise or aspirin intake enhanced the allergic reaction onset as cofactors. The prevalence of GRP sensitization was high in Japanese fruit allergy patients except for PFAS patients. In conclusion, GRP-sensitized patients may have allergies to multiple fruits and may show peculiar characteristics such as facial swelling and cofactor dependence. © 2017 Japanese Dermatological Association.

  1. A disease-causing mutation illuminates the protein membrane topology of the kidney-expressed prohibitin homology (PHB) domain protein podocin.

    Science.gov (United States)

    Schurek, Eva-Maria; Völker, Linus A; Tax, Judit; Lamkemeyer, Tobias; Rinschen, Markus M; Ungrue, Denise; Kratz, John E; Sirianant, Lalida; Kunzelmann, Karl; Chalfie, Martin; Schermer, Bernhard; Benzing, Thomas; Höhne, Martin

    2014-04-18

    Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.

  2. New acute transforming feline retovirus with fms homology specifies a C-terminally truncated version of the c-fms protein that is different from SM-feline sarcoma virus v-fms protein

    International Nuclear Information System (INIS)

    Besmer, P.; Lader, E.; George, P.C.; Bergold, P.J.; Qui, F.; Zuckerman, E.E.; Hardy, W.D.

    1986-01-01

    The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of the SM-FeSV. The genome organization of the 8.6-kilobase HZ5-FeSV provirus is 5' Δgag-fms-Δpol-Δenv 3'. The HZ5- and SM-FeSVs display indistinguishable in vitro transformation characteristics, and the structures of the gag-fms transforming genes in the two viruses are very similar. In the HZ5-FeSV and the SM-FeSV, identical c-fms and feline leukemia virus p10 sequences form the 5' gag-fms junction. With regard to v-fms the two viruses are homologous up to 11 amino acids before the C terminus of the SM-FeSV v-fms protein. In HZ5-FeSV a segment of 362 nucleotides then follows before the 3' recombination site with feline leukemia virus pol. The new 3' v-fms sequence encodes 27 amino acids before reaching a TGA termination signal. The relationship of this sequence with the recently characterized human c-fms sequence has been examined. The 3' HZ5-FeSV v-fms sequence is homologous with 3' c-fms sequences. A frameshift mutation (11-base-pair deletion) was found in the C-terminal fms coding sequence of the HZ5-FeSV. As a result, the HZ5-FeSV v-fms protein is predicted to be a C-terminally truncated version of c-fms. This frameshift mutation may determine the oncogenic properties of v-fms in the HZ5-FeSV

  3. New acute transforming feline retovirus with fms homology specifies a C-terminally truncated version of the c-fms protein that is different from SM-feline sarcoma virus v-fms protein

    Energy Technology Data Exchange (ETDEWEB)

    Besmer, P.; Lader, E.; George, P.C.; Bergold, P.J.; Qui, F.; Zuckerman, E.E.; Hardy, W.D.

    1986-10-01

    The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of the SM-FeSV. The genome organization of the 8.6-kilobase HZ5-FeSV provirus is 5' ..delta..gag-fms-..delta..pol-..delta..env 3'. The HZ5- and SM-FeSVs display indistinguishable in vitro transformation characteristics, and the structures of the gag-fms transforming genes in the two viruses are very similar. In the HZ5-FeSV and the SM-FeSV, identical c-fms and feline leukemia virus p10 sequences form the 5' gag-fms junction. With regard to v-fms the two viruses are homologous up to 11 amino acids before the C terminus of the SM-FeSV v-fms protein. In HZ5-FeSV a segment of 362 nucleotides then follows before the 3' recombination site with feline leukemia virus pol. The new 3' v-fms sequence encodes 27 amino acids before reaching a TGA termination signal. The relationship of this sequence with the recently characterized human c-fms sequence has been examined. The 3' HZ5-FeSV v-fms sequence is homologous with 3' c-fms sequences. A frameshift mutation (11-base-pair deletion) was found in the C-terminal fms coding sequence of the HZ5-FeSV. As a result, the HZ5-FeSV v-fms protein is predicted to be a C-terminally truncated version of c-fms. This frameshift mutation may determine the oncogenic properties of v-fms in the HZ5-FeSV.

  4. Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp

    Directory of Open Access Journals (Sweden)

    Devendra K. Rai

    2012-07-01

    Full Text Available Bovine Rhinitis B Virus (BRBV is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp, which is also known as 3D polymerase (3Dpol. In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P, nucleotide tri-phosphate (NTP and pyrophosphate (PPi bound FMDV 3Dpol (PDB, 1U09 and 2E9Z. The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16 and rabbit hemorrhagic disease virus (RHDV 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the

  5. Homology in Electromagnetic Boundary Value Problems

    Directory of Open Access Journals (Sweden)

    Pellikka Matti

    2010-01-01

    Full Text Available We discuss how homology computation can be exploited in computational electromagnetism. We represent various cellular mesh reduction techniques, which enable the computation of generators of homology spaces in an acceptable time. Furthermore, we show how the generators can be used for setting up and analysis of an electromagnetic boundary value problem. The aim is to provide a rationale for homology computation in electromagnetic modeling software.

  6. Protein secondary structure prediction for a single-sequence using hidden semi-Markov models

    Directory of Open Access Journals (Sweden)

    Borodovsky Mark

    2006-03-01

    Full Text Available Abstract Background The accuracy of protein secondary structure prediction has been improving steadily towards the 88% estimated theoretical limit. There are two types of prediction algorithms: Single-sequence prediction algorithms imply that information about other (homologous proteins is not available, while algorithms of the second type imply that information about homologous proteins is available, and use it intensively. The single-sequence algorithms could make an important contribution to studies of proteins with no detected homologs, however the accuracy of protein secondary structure prediction from a single-sequence is not as high as when the additional evolutionary information is present. Results In this paper, we further refine and extend the hidden semi-Markov model (HSMM initially considered in the BSPSS algorithm. We introduce an improved residue dependency model by considering the patterns of statistically significant amino acid correlation at structural segment borders. We also derive models that specialize on different sections of the dependency structure and incorporate them into HSMM. In addition, we implement an iterative training method to refine estimates of HSMM parameters. The three-state-per-residue accuracy and other accuracy measures of the new method, IPSSP, are shown to be comparable or better than ones for BSPSS as well as for PSIPRED, tested under the single-sequence condition. Conclusions We have shown that new dependency models and training methods bring further improvements to single-sequence protein secondary structure prediction. The results are obtained under cross-validation conditions using a dataset with no pair of sequences having significant sequence similarity. As new sequences are added to the database it is possible to augment the dependency structure and obtain even higher accuracy. Current and future advances should contribute to the improvement of function prediction for orphan proteins inscrutable

  7. Lectures on functor homology

    CERN Document Server

    Touzé, Antoine

    2015-01-01

    This book features a series of lectures that explores three different fields in which functor homology (short for homological algebra in functor categories) has recently played a significant role. For each of these applications, the functor viewpoint provides both essential insights and new methods for tackling difficult mathematical problems. In the lectures by Aurélien Djament, polynomial functors appear as coefficients in the homology of infinite families of classical groups, e.g. general linear groups or symplectic groups, and their stabilization. Djament’s theorem states that this stable homology can be computed using only the homology with trivial coefficients and the manageable functor homology. The series includes an intriguing development of Scorichenko’s unpublished results. The lectures by Wilberd van der Kallen lead to the solution of the general cohomological finite generation problem, extending Hilbert’s fourteenth problem and its solution to the context of cohomology. The focus here is o...

  8. Protein structure modelling and evaluation based on a 4-distance description of side-chain interactions

    Directory of Open Access Journals (Sweden)

    Inbar Yuval

    2010-07-01

    Full Text Available Abstract Background Accurate evaluation and modelling of residue-residue interactions within and between proteins is a key aspect of computational structure prediction including homology modelling, protein-protein docking, refinement of low-resolution structures, and computational protein design. Results Here we introduce a method for accurate protein structure modelling and evaluation based on a novel 4-distance description of residue-residue interaction geometry. Statistical 4-distance preferences were extracted from high-resolution protein structures and were used as a basis for a knowledge-based potential, called Hunter. We demonstrate that 4-distance description of side chain interactions can be used reliably to discriminate the native structure from a set of decoys. Hunter ranked the native structure as the top one in 217 out of 220 high-resolution decoy sets, in 25 out of 28 "Decoys 'R' Us" decoy sets and in 24 out of 27 high-resolution CASP7/8 decoy sets. The same concept was applied to side chain modelling in protein structures. On a set of very high-resolution protein structures the average RMSD was 1.47 Å for all residues and 0.73 Å for buried residues, which is in the range of attainable accuracy for a model. Finally, we show that Hunter performs as good or better than other top methods in homology modelling based on results from the CASP7 experiment. The supporting web site http://bioinfo.weizmann.ac.il/hunter/ was developed to enable the use of Hunter and for visualization and interactive exploration of 4-distance distributions. Conclusions Our results suggest that Hunter can be used as a tool for evaluation and for accurate modelling of residue-residue interactions in protein structures. The same methodology is applicable to other areas involving high-resolution modelling of biomolecules.

  9. Modeling of allergen proteins found in sea food products

    Directory of Open Access Journals (Sweden)

    Nataly Galán-Freyle

    2012-06-01

    Full Text Available Shellfish are a source of food allergens, and their consumption is the cause of severe allergic reactions in humans. Tropomyosins, a family of muscle proteins, have been identified as the major allergens in shellfish and mollusks species. Nevertheless, few experimentally determined three-dimensional structures are available in the Protein Data Base (PDB. In this study, 3D models of several homologous of tropomyosins present in marine shellfish and mollusk species (Chaf 1, Met e1, Hom a1, Per v1, and Pen a1 were constructed, validated, and their immunoglobulin E binding epitopes were identified using bioinformatics tools. All protein models for these allergens consisted of long alpha-helices. Chaf 1, Met e1, and Hom a1 had six conserved regions with sequence similarities to known epitopes, whereas Per v1 and Pen a1 contained only one. Lipophilic potentials of identified epitopes revealed a high propensity of hydrophobic amino acids in the immunoglobulin E binding site. This information could be useful to design tropomyosin-specific immunotherapy for sea food allergies.

  10. An HMM posterior decoder for sequence feature prediction that includes homology information

    DEFF Research Database (Denmark)

    Käll, Lukas; Krogh, Anders Stærmose; Sonnhammer, Erik L. L.

    2005-01-01

    Motivation: When predicting sequence features like transmembrane topology, signal peptides, coil-coil structures, protein secondary structure or genes, extra support can be gained from homologs. Results: We present here a general hidden Markov model (HMM) decoding algorithm that combines probabil......Motivation: When predicting sequence features like transmembrane topology, signal peptides, coil-coil structures, protein secondary structure or genes, extra support can be gained from homologs. Results: We present here a general hidden Markov model (HMM) decoding algorithm that combines......://phobius.cgb.ki.se/poly.html . An implementation of the algorithm is available on request from the authors....

  11. Homological stabilizer codes

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Jonas T., E-mail: jonastyleranderson@gmail.com

    2013-03-15

    In this paper we define homological stabilizer codes on qubits which encompass codes such as Kitaev's toric code and the topological color codes. These codes are defined solely by the graphs they reside on. This feature allows us to use properties of topological graph theory to determine the graphs which are suitable as homological stabilizer codes. We then show that all toric codes are equivalent to homological stabilizer codes on 4-valent graphs. We show that the topological color codes and toric codes correspond to two distinct classes of graphs. We define the notion of label set equivalencies and show that under a small set of constraints the only homological stabilizer codes without local logical operators are equivalent to Kitaev's toric code or to the topological color codes. - Highlights: Black-Right-Pointing-Pointer We show that Kitaev's toric codes are equivalent to homological stabilizer codes on 4-valent graphs. Black-Right-Pointing-Pointer We show that toric codes and color codes correspond to homological stabilizer codes on distinct graphs. Black-Right-Pointing-Pointer We find and classify all 2D homological stabilizer codes. Black-Right-Pointing-Pointer We find optimal codes among the homological stabilizer codes.

  12. A sensitive short read homology search tool for paired-end read sequencing data.

    Science.gov (United States)

    Techa-Angkoon, Prapaporn; Sun, Yanni; Lei, Jikai

    2017-10-16

    Homology search is still a significant step in functional analysis for genomic data. Profile Hidden Markov Model-based homology search has been widely used in protein domain analysis in many different species. In particular, with the fast accumulation of transcriptomic data of non-model species and metagenomic data, profile homology search is widely adopted in integrated pipelines for functional analysis. While the state-of-the-art tool HMMER has achieved high sensitivity and accuracy in domain annotation, the sensitivity of HMMER on short reads declines rapidly. The low sensitivity on short read homology search can lead to inaccurate domain composition and abundance computation. Our experimental results showed that half of the reads were missed by HMMER for a RNA-Seq dataset. Thus, there is a need for better methods to improve the homology search performance for short reads. We introduce a profile homology search tool named Short-Pair that is designed for short paired-end reads. By using an approximate Bayesian approach employing distribution of fragment lengths and alignment scores, Short-Pair can retrieve the missing end and determine true domains. In particular, Short-Pair increases the accuracy in aligning short reads that are part of remote homologs. We applied Short-Pair to a RNA-Seq dataset and a metagenomic dataset and quantified its sensitivity and accuracy on homology search. The experimental results show that Short-Pair can achieve better overall performance than the state-of-the-art methodology of profile homology search. Short-Pair is best used for next-generation sequencing (NGS) data that lack reference genomes. It provides a complementary paired-end read homology search tool to HMMER. The source code is freely available at https://sourceforge.net/projects/short-pair/ .

  13. Protein modelling of triterpene synthase genes from mangrove plants using Phyre2 and Swiss-model

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sulistiyono, N.; Hayati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

    2018-03-01

    Molecular cloning of five oxidosqualene cyclases (OSC) genes from Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa had previously been cloned, characterized, and encoded mono and -multi triterpene synthases. The present study analyzed protein modelling of triterpene synthase genes from mangrove using Phyre2 and Swiss-model. The diversity was noted within protein modelling of triterpene synthases using Phyre2 from sequence identity (38-43%) and residue (696-703). RsM2 was distinguishable from others for template structure; it used lanosterol synthase as a template (PDB ID: w6j.1.A). By contrast, other genes used human lanosterol synthase (1w6k.1.A). The predicted bind sites were correlated with the product of triterpene synthase, the product of BgbAS was β-amyrin, while RsM1 contained a significant amount of β-amyrin. Similarly BgLUS and KcMS, both main products was lupeol, on the other hand, RsM2 with the outcome of taraxerol. Homology modelling revealed that 696 residues of BgbAS, BgLUS, RsM1, and RsM2 (91-92% of the amino acid sequence) had been modelled with 100% confidence by the single highest scoring template using Phyre2. This coverage was higher than Swiss-model (85-90%). The present study suggested that molecular cloning of triterpene genes provides useful tools for studying the protein modelling related regulation of isoprenoids biosynthesis in mangrove forests.

  14. GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Bohmdorfer, G.; Schleiffer, A.; Brunmeir, R.; Ferscha, S.; Nizhynska, V.; Kozák, Jaroslav; Angelis, Karel; Kreil, D. P.; Schweizer, D.

    2011-01-01

    Roč. 67, č. 3 (2011), s. 420-433 ISSN 0960-7412 R&D Projects: GA MŠk 1M0505; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : structural maintenance of chromosomes * DNA repair * somatic homologous recombination Subject RIV: EI - Biotechnology ; Bionics Impact factor: 6.160, year: 2011

  15. Patterning of inflorescences and flowers by the F-Box protein DOUBLE TOP and the LEAFY homolog ABERRANT LEAF AND FLOWER of petunia.

    Science.gov (United States)

    Souer, Erik; Rebocho, Alexandra B; Bliek, Mattijs; Kusters, Elske; de Bruin, Robert A M; Koes, Ronald

    2008-08-01

    Angiosperms display a wide variety of inflorescence architectures differing in the positions where flowers or branches arise. The expression of floral meristem identity (FMI) genes determines when and where flowers are formed. In Arabidopsis thaliana, this is regulated via transcription of LEAFY (LFY), which encodes a transcription factor that promotes FMI. We found that this is regulated in petunia (Petunia hybrida) via transcription of a distinct gene, DOUBLE TOP (DOT), a homolog of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Mutation of DOT or its tomato (Solanum lycopersicum) homolog ANANTHA abolishes FMI. Ubiquitous expression of DOT or UFO in petunia causes very early flowering and transforms the inflorescence into a solitary flower and leaves into petals. Ectopic expression of DOT or UFO together with LFY or its homolog ABERRANT LEAF AND FLOWER (ALF) in petunia seedlings activates genes required for identity or outgrowth of organ primordia. DOT interacts physically with ALF, suggesting that it activates ALF by a posttranslational mechanism. Our findings suggest a wider role than previously thought for DOT and UFO in the patterning of flowers and indicate that the different roles of LFY and UFO homologs in the spatiotemporal control of floral identity in distinct species result from their divergent expression patterns.

  16. Pyrid-2-yl and 2-CyanoPhenyl fused heterocyclic compounds as human P2X3 inhibitors: a combined approach based on homology modelling, docking and QSAR analysis.

    Science.gov (United States)

    Janardhan, Sridhara; Seth, Subhendu; Viswanadhan, Vellarkad N

    2014-02-01

    P2X receptors are hetero-oligomeric proteins that function as membrane ion channels and are gated by extracellular ATP. The hP2X[Formula: see text] subunit is a constituent of the channels on a subset of sensory neurons involved in pain signaling, where ATP released by damaged and inflamed tissue can initiate action potentials. Hence, the inhibition of ATP-activated P2X3 receptor is an exciting approach for the treatment of inflammatory and neuropathic pain. Recently, the crystal structures of zebrafish P2X4 (zP2X4) were obtained in closed, apo state (PDB ID: 3I5D) and ATP-bound, open state (PDB ID: 4DW1). These structures were used to develop a homology model of human P2X3 (hP2X3 in order to identify through docking studies, the binding modes of known P2X3 inhibitors and their key active site interactions, along with a pharmacophore-based 3D-QSAR model for a series of 136 Pyrid-2-yl and 2-CyanoPhenyl fused heterocyclic compounds. These 3D-QSAR models have been developed with different combinations of training and test set divisions obtained by random separation, Jarvis-Patrick clustering, K-means clustering and sphere exclusion methods. The best predictive 3D-QSAR model resulted in training set R2 of 0.75, internal test set Q2 of 0.74, Pearson-R value of 0.87 and root mean square error of 0.37. The information generated by the pharmacophore model and docking analyses using the homology model provides valuable clues to design novel potent hP2X3 inhibitors.

  17. Predicting turns in proteins with a unified model.

    Directory of Open Access Journals (Sweden)

    Qi Song

    Full Text Available MOTIVATION: Turns are a critical element of the structure of a protein; turns play a crucial role in loops, folds, and interactions. Current prediction methods are well developed for the prediction of individual turn types, including α-turn, β-turn, and γ-turn, etc. However, for further protein structure and function prediction it is necessary to develop a uniform model that can accurately predict all types of turns simultaneously. RESULTS: In this study, we present a novel approach, TurnP, which offers the ability to investigate all the turns in a protein based on a unified model. The main characteristics of TurnP are: (i using newly exploited features of structural evolution information (secondary structure and shape string of protein based on structure homologies, (ii considering all types of turns in a unified model, and (iii practical capability of accurate prediction of all turns simultaneously for a query. TurnP utilizes predicted secondary structures and predicted shape strings, both of which have greater accuracy, based on innovative technologies which were both developed by our group. Then, sequence and structural evolution features, which are profile of sequence, profile of secondary structures and profile of shape strings are generated by sequence and structure alignment. When TurnP was validated on a non-redundant dataset (4,107 entries by five-fold cross-validation, we achieved an accuracy of 88.8% and a sensitivity of 71.8%, which exceeded the most state-of-the-art predictors of certain type of turn. Newly determined sequences, the EVA and CASP9 datasets were used as independent tests and the results we achieved were outstanding for turn predictions and confirmed the good performance of TurnP for practical applications.

  18. Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

    International Nuclear Information System (INIS)

    Cui Jianzhou; Shen Xueyan; Yan Zuowei; Zhao Haobin; Nagahama, Yoshitaka

    2009-01-01

    Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meERα and huERα, meERβ1 and huERβ, meERβ2, and huERβ with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meARα and huAR, meARβ, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for Cα atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.

  19. New insight in the structural features of haloadaptation in α-amylases from halophilic Archaea following homology modeling strategy: folded and stable conformation maintained through low hydrophobicity and highly negative charged surface

    Science.gov (United States)

    Zorgani, Mohamed Amine; Patron, Kevin; Desvaux, Mickaël

    2014-07-01

    Proteins from halophilic archaea, which live in extreme saline conditions, have evolved to remain folded, active and stable at very high ionic strengths. Understanding the mechanism of haloadaptation is the first step toward engineering of halostable biomolecules. Amylases are one of the main enzymes used in industry. Yet, no three-dimensional structure has been experimentally resolved for α-amylases from halophilic archaea. In this study, homology structure modeling of α-amylases from the halophilic archaea Haloarcula marismortui, Haloarcula hispanica, and Halalkalicoccus jeotgali were performed. The resulting models were subjected to energy minimization, evaluation, and structural analysis. Calculations of the amino acid composition, salt bridges and hydrophobic interactions were also performed and compared to a set of non-halophilic counterparts. It clearly appeared that haloarchaeal α-amylases exhibited lower propensities for helix formation and higher propensities for coil-forming regions. Furthermore, they could maintain a folded and stable conformation in high salt concentration through highly negative charged surface with over representation of acidic residues, especially Asp, and low hydrophobicity with increase of salt bridges and decrease in hydrophobic interactions on the protein surface. This study sheds some light on the stability of α-amylases from halophilic archaea and provides strong basis not only to understand haloadaptation mechanisms of proteins in microorganisms from hypersalines environments but also for biotechnological applications.

  20. The PAS fold: A redefinition of the PAS domain based upon structural prediction. A large-scale homology modelling study

    NARCIS (Netherlands)

    Hefti, M.H.; Francoijs, C.J.J.; Vries, de S.C.; Dixon, R.; Vervoort, J.J.M.

    2004-01-01

    In the postgenomic era it is essential that protein sequences are annotated correctly in order to help in the assignment of their putative functions. Over 1300 proteins in current protein sequence databases are predicted to contain a PAS domain based upon amino acid sequence alignments. One of the

  1. Geometric homology revisited

    OpenAIRE

    Ruffino, Fabio Ferrari

    2013-01-01

    Given a cohomology theory, there is a well-known abstract way to define the dual homology theory using the theory of spectra. In [4] the author provides a more geometric construction of the homology theory, using a generalization of the bordism groups. Such a generalization involves in its definition the vector bundle modification, which is a particular case of the Gysin map. In this paper we provide a more natural variant of that construction, which replaces the vector bundle modification wi...

  2. Homology-based Modeling of Rhodopsin-like Family Members in the Inactive State: Structural Analysis and Deduction of Tips for Modeling and Optimization.

    Science.gov (United States)

    Pappalardo, Matteo; Rayan, Mahmoud; Abu-Lafi, Saleh; Leonardi, Martha E; Milardi, Danilo; Guccione, Salvatore; Rayan, Anwar

    2017-08-01

    Modeling G-Protein Coupled Receptors (GPCRs) is an emergent field of research, since utility of high-quality models in receptor structure-based strategies might facilitate the discovery of interesting drug candidates. The findings from a quantitative analysis of eighteen resolved structures of rhodopsin family "A" receptors crystallized with antagonists and 153 pairs of structures are described. A strategy termed endeca-amino acids fragmentation was used to analyze the structures models aiming to detect the relationship between sequence identity and Root Mean Square Deviation (RMSD) at each trans-membrane-domain. Moreover, we have applied the leave-one-out strategy to study the shiftiness likelihood of the helices. The type of correlation between sequence identity and RMSD was studied using the aforementioned set receptors as representatives of membrane proteins and 98 serine proteases with 4753 pairs of structures as representatives of globular proteins. Data analysis using fragmentation strategy revealed that there is some extent of correlation between sequence identity and global RMSD of 11AA width windows. However, spatial conservation is not always close to the endoplasmic side as was reported before. A comparative study with globular proteins shows that GPCRs have higher standard deviation and higher slope in the graph with correlation between sequence identity and RMSD. The extracted information disclosed in this paper could be incorporated in the modeling protocols while using technique for model optimization and refinement. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Competition between replicative and translesion polymerases during homologous recombination repair in Drosophila.

    Directory of Open Access Journals (Sweden)

    Daniel P Kane

    Full Text Available In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis.

  4. Metagenomic identification of a novel salt tolerance gene from the human gut microbiome which encodes a membrane protein with homology to a brp/blh-family β-carotene 15,15'-monooxygenase.

    Directory of Open Access Journals (Sweden)

    Eamonn P Culligan

    Full Text Available The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.

  5. Arabidopsis KHZ1 and KHZ2, two novel non-tandem CCCH zinc-finger and K-homolog domain proteins, have redundant roles in the regulation of flowering and senescence.

    Science.gov (United States)

    Yan, Zongyun; Jia, Jianheng; Yan, Xiaoyuan; Shi, Huiying; Han, Yuzhen

    2017-12-01

    The two novel CCCH zinc-finger and K-homolog (KH) proteins, KHZ1 and KHZ2, play important roles in regulating flowering and senescence redundantly in Arabidopsis. The CCCH zinc-finger proteins and K-homolog (KH) proteins play important roles in plant development and stress responses. However, the biological functions of many CCCH zinc-finger proteins and KH proteins remain uncharacterized. In Arabidopsis, KHZ1 and KHZ2 are characterized as two novel CCCH zinc-finger and KH domain proteins which belong to subfamily VII in CCCH family. We obtained khz1, khz2 mutants and khz1 khz2 double mutants, as well as overexpression (OE) lines of KHZ1 and KHZ2. Compared with the wild type (WT), the khz2 mutants displayed no defects in growth and development, and the khz1 mutants were slightly late flowering, whereas the khz1 khz2 double mutants showed a pronounced late flowering phenotype. In contrast, artificially overexpressing KHZ1 and KHZ2 led to the early flowering. Consistent with the late flowering phenotype, the expression of flowering repressor gene FLC was up-regulated, while the expression of flowering integrator and floral meristem identity (FMI) genes were down-regulated significantly in khz1 khz2. In addition, we also observed that the OE plants of KHZ1 and KHZ2 showed early leaf senescence significantly, whereas the khz1 khz2 double mutants showed delayed senescence of leaf and the whole plant. Both KHZ1 and KHZ2 were ubiquitously expressed throughout the tissues of Arabidopsis. KHZ1 and KHZ2 were localized to the nucleus, and possessed both transactivation activities and RNA-binding abilities. Taken together, we conclude that KHZ1 and KHZ2 have redundant roles in the regulation of flowering and senescence in Arabidopsis.

  6. A member of a new plant gene family encoding a meprin and TRAF homology (MATH) domain-containing protein is involved in restriction of long distance movement of plant viruses

    Science.gov (United States)

    Cosson, Patrick; Sofer, Luc; Schurdi-Levraud, Valérie

    2010-01-01

    Restriction of long distance movement of several potyviruses in Arabidopsis thaliana is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2 and RTM3 and acts as a non-conventional resistance. RTM1 encodes a protein belonging to the jacalin family and RTM2 encodes a protein which has similarities to small heat shock proteins. The recent cloning of RTM3 which encodes a protein belonging to an unknown protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its N-terminal region and a coiled-coil (CC) domain at its C-terminal end is an important breakthrough for a better understanding of this resistance process. Not only the third gene involved in this resistance has been identified and has allowed revealing a new gene family in plant but the discovery that the RTM3 protein interacts directly with RTM1 strongly suggests that the RTM proteins form a multimeric complex. However, these data also highlight striking similarities of the RTM resistance with the well known R-gene mediated resistance. PMID:20930558

  7. Phylogenetic analysis of the diacylglycerol kinase family of proteins and identification of multiple highly-specific conserved inserts and deletions within the catalytic domain that are distinctive characteristics of different classes of DGK homologs.

    Directory of Open Access Journals (Sweden)

    Radhey S Gupta

    Full Text Available Diacylglycerol kinase (DGK family of proteins, which phosphorylates diacylglycerol into phosphatidic acid, play important role in controlling diverse cellular processes in eukaryotic organisms. Most vertebrate species contain 10 different DGK isozymes, which are grouped into 5 different classes based on the presence or absence of specific functional domains. However, the relationships among different DGK isozymes or how they have evolved from a common ancestor is unclear. The catalytic domain constitutes the single largest sequence element within the DGK proteins that is commonly and uniquely shared by all family members, but there is limited understanding of the overall function of this domain. In this work, we have used the catalytic domain sequences to construct a phylogenetic tree for the DGK family members from representatives of the main vertebrate classes and have also examined the distributions of various DGK isozymes in eukaryotic phyla. In a tree based on catalytic domain sequences, the DGK homologs belonging to different classes formed strongly supported clusters which were separated by long branches, and the different isozymes within each class also generally formed monophyletic groupings. Further, our analysis of the sequence alignments of catalytic domains has identified >10 novel sequence signatures consisting of conserved signature indels (inserts or deletions, CSIs that are distinctive characteristics of either particular classes of DGK isozymes, or are commonly shared by members of two or more classes of DGK isozymes. The conserved indels in protein sequences are known to play important functional roles in the proteins/organisms where they are found. Thus, our identification of multiple highly specific CSIs that are distinguishing characteristics of different classes of DGK homologs points to the existence of important differences in the catalytic domain function among the DGK isozymes. The identified CSIs in conjunction with

  8. Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kitagaki, Hiroshi; Wu, Hong; Shimoi, Hitoshi; Ito, Kiyoshi

    2002-11-01

    The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.

  9. Discovery of rare protein-coding genes in model methylotroph Methylobacterium extorquens AM1.

    Science.gov (United States)

    Kumar, Dhirendra; Mondal, Anupam Kumar; Yadav, Amit Kumar; Dash, Debasis

    2014-12-01

    Proteogenomics involves the use of MS to refine annotation of protein-coding genes and discover genes in a genome. We carried out comprehensive proteogenomic analysis of Methylobacterium extorquens AM1 (ME-AM1) from publicly available proteomics data with a motive to improve annotation for methylotrophs; organisms capable of surviving in reduced carbon compounds such as methanol. Besides identifying 2482(50%) proteins, 29 new genes were discovered and 66 annotated gene models were revised in ME-AM1 genome. One such novel gene is identified with 75 peptides, lacks homolog in other methylobacteria but has glycosyl transferase and lipopolysaccharide biosynthesis protein domains, indicating its potential role in outer membrane synthesis. Many novel genes are present only in ME-AM1 among methylobacteria. Distant homologs of these genes in unrelated taxonomic classes and low GC-content of few genes suggest lateral gene transfer as a potential mode of their origin. Annotations of methylotrophy related genes were also improved by the discovery of a short gene in methylotrophy gene island and redefining a gene important for pyrroquinoline quinone synthesis, essential for methylotrophy. The combined use of proteogenomics and rigorous bioinformatics analysis greatly enhanced the annotation of protein-coding genes in model methylotroph ME-AM1 genome. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Structural model of dodecameric heat-shock protein Hsp21

    DEFF Research Database (Denmark)

    Rutsdottir, Gudrun; Härmark, Johan; Weide, Yoran

    2017-01-01

    for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer...

  11. Three-dimensional models of Mycobacterium tuberculosis proteins Rv1555, Rv1554 and their docking analyses with sildenafil, tadalafil, vardenafil drugs, suggest interference with quinol binding likely to affect protein's function.

    Science.gov (United States)

    Dash, Pallabini; Bala Divya, M; Guruprasad, Lalitha; Guruprasad, Kunchur

    2018-04-18

    Earlier based on bioinformatics analyses, we had predicted the Mycobacterium tuberculosis (M.tb) proteins; Rv1555 and Rv1554, among the potential new tuberculosis drug targets. According to the 'TB-drugome' the Rv1555 protein is 'druggable' with sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra) drugs. In the present work, we intended to understand via computer modeling studies, how the above drugs are likely to inhibit the M.tb protein's function. The three-dimensional computer models for M.tb proteins; Rv1555 and Rv1554 constructed on the template of equivalent membrane anchor subunits of the homologous E.coli quinol fumarate reductase respiratory protein complex, followed by drug docking analyses, suggested that the binding of above drugs interferes with quinol binding sites. Also, we experimentally observed the in-vitro growth inhibition of E.coli bacteria containing the homologous M.tb protein sequences with sildenafil and tadalafil drugs. The predicted binding sites of the drugs is likely to affect the above M.tb proteins function as quinol binding is known to be essential for electron transfer function during anaerobic respiration in the homologous E.coli protein complex. Therefore, sildenafil and related drugs currently used in the treatment of male erectile dysfunction targeting the human phosphodiesterase 5 enzyme may be evaluated for their plausible role as repurposed drugs to treat human tuberculosis.

  12. Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB.

    Science.gov (United States)

    Kemege, Kyle E; Hickey, John M; Barta, Michael L; Wickstrum, Jason; Balwalli, Namita; Lovell, Scott; Battaile, Kevin P; Hefty, P Scott

    2015-02-01

    Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient Escherichia coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia. © 2014 John Wiley & Sons Ltd.

  13. Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

    Science.gov (United States)

    Kemege, Kyle E.; Hickey, John M.; Barta, Michael L.; Wickstrum, Jason; Balwalli, Namita; Lovell, Scott; Battaile, Kevin P.; Hefty, P. Scott

    2015-01-01

    Summary Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis, and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient E. coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia. PMID:25382739

  14. Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment

    KAUST Repository

    Lensink, Marc F.

    2016-04-28

    We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. © 2016 Wiley Periodicals, Inc.

  15. Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment

    KAUST Repository

    Lensink, Marc F.; Velankar, Sameer; Kryshtafovych, Andriy; Huang, Shen-You; Schneidman-Duhovny, Dina; Sali, Andrej; Segura, Joan; Fernandez-Fuentes, Narcis; Viswanath, Shruthi; Elber, Ron; Grudinin, Sergei; Popov, Petr; Neveu, Emilie; Lee, Hasup; Baek, Minkyung; Park, Sangwoo; Heo, Lim; Rie Lee, Gyu; Seok, Chaok; Qin, Sanbo; Zhou, Huan-Xiang; Ritchie, David W.; Maigret, Bernard; Devignes, Marie-Dominique; Ghoorah, Anisah; Torchala, Mieczyslaw; Chaleil, Raphaë l A.G.; Bates, Paul A.; Ben-Zeev, Efrat; Eisenstein, Miriam; Negi, Surendra S.; Weng, Zhiping; Vreven, Thom; Pierce, Brian G.; Borrman, Tyler M.; Yu, Jinchao; Ochsenbein, Franç oise; Guerois, Raphaë l; Vangone, Anna; Rodrigues, Joã o P.G.L.M.; van Zundert, Gydo; Nellen, Mehdi; Xue, Li; Karaca, Ezgi; Melquiond, Adrien S.J.; Visscher, Koen; Kastritis, Panagiotis L.; Bonvin, Alexandre M.J.J.; Xu, Xianjin; Qiu, Liming; Yan, Chengfei; Li, Jilong; Ma, Zhiwei; Cheng, Jianlin; Zou, Xiaoqin; Shen, Yang; Peterson, Lenna X.; Kim, Hyung-Rae; Roy, Amit; Han, Xusi; Esquivel-Rodriguez, Juan; Kihara, Daisuke; Yu, Xiaofeng; Bruce, Neil J.; Fuller, Jonathan C.; Wade, Rebecca C.; Anishchenko, Ivan; Kundrotas, Petras J.; Vakser, Ilya A.; Imai, Kenichiro; Yamada, Kazunori; Oda, Toshiyuki; Nakamura, Tsukasa; Tomii, Kentaro; Pallara, Chiara; Romero-Durana, Miguel; Jimé nez-Garcí a, Brian; Moal, Iain H.; Fé rnandez-Recio, Juan; Joung, Jong Young; Kim, Jong Yun; Joo, Keehyoung; Lee, Jooyoung; Kozakov, Dima; Vajda, Sandor; Mottarella, Scott; Hall, David R.; Beglov, Dmitri; Mamonov, Artem; Xia, Bing; Bohnuud, Tanggis; Del Carpio, Carlos A.; Ichiishi, Eichiro; Marze, Nicholas; Kuroda, Daisuke; Roy Burman, Shourya S.; Gray, Jeffrey J.; Chermak, Edrisse; Cavallo, Luigi; Oliva, Romina; Tovchigrechko, Andrey; Wodak, Shoshana J.

    2016-01-01

    We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. © 2016 Wiley Periodicals, Inc.

  16. A simple recipe for the non-expert bioinformaticist for building experimentally-testable hypotheses for proteins with no known homologs

    CSIR Research Space (South Africa)

    Zawaira, A

    2012-12-01

    Full Text Available The study of the protein-protein interactions (PPIs) of unique ORFs is a strategy for deciphering the biological roles of unique ORFs of interest. For uniform reference, we define unique ORFs as those for which no matching protein is found after PDB...

  17. Crystallization and preliminary crystallographic analysis of the human calcineurin homologous protein CHP2 bound to the cytoplasmic region of the Na{sup +}/H{sup +} exchanger NHE1

    Energy Technology Data Exchange (ETDEWEB)

    Ben Ammar, Youssef [Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Takeda, Soichi [Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Sugawara, Mitsuaki; Miyano, Masashi [Structural Biophysics Laboratory, RIKEN Harima Institute at SPring-8, Kouto, Mikazuki, Sayo, Hyogo 679-5148 (Japan); Mori, Hidezo [Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Wakabayashi, Shigeo, E-mail: wak@ri.ncvc.go.jp [Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan)

    2005-10-01

    Crystallization of the human CHP2–NHE1 binding domain complex. Calcineurin homologous protein (CHP) is a Ca{sup 2+}-binding protein that directly interacts with and regulates the activity of all plasma-membrane Na{sup +}/H{sup +}-exchanger (NHE) family members. In contrast to the ubiquitous isoform CHP1, CHP2 is highly expressed in cancer cells. To understand the regulatory mechanism of NHE1 by CHP2, the complex CHP2–NHE1 (amino acids 503–545) has been crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as precipitant. The crystals diffract to 2.7 Å and belong to a tetragonal space group, with unit-cell parameters a = b = 49.96, c = 103.20 Å.

  18. Physiologically Based Pharmacokinetic Modeling of Therapeutic Proteins.

    Science.gov (United States)

    Wong, Harvey; Chow, Timothy W

    2017-09-01

    Biologics or therapeutic proteins are becoming increasingly important as treatments for disease. The most common class of biologics are monoclonal antibodies (mAbs). Recently, there has been an increase in the use of physiologically based pharmacokinetic (PBPK) modeling in the pharmaceutical industry in drug development. We review PBPK models for therapeutic proteins with an emphasis on mAbs. Due to their size and similarity to endogenous antibodies, there are distinct differences between PBPK models for small molecules and mAbs. The high-level organization of a typical mAb PBPK model consists of a whole-body PBPK model with organ compartments interconnected by both blood and lymph flows. The whole-body PBPK model is coupled with tissue-level submodels used to describe key mechanisms governing mAb disposition including tissue efflux via the lymphatic system, elimination by catabolism, protection from catabolism binding to the neonatal Fc (FcRn) receptor, and nonlinear binding to specific pharmacological targets of interest. The use of PBPK modeling in the development of therapeutic proteins is still in its infancy. Further application of PBPK modeling for therapeutic proteins will help to define its developing role in drug discovery and development. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  19. A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

    Science.gov (United States)

    Donnini, Sandra; Finetti, Federica; Francese, Simona; Boscaro, Francesca; Dani, Francesca R; Maset, Fabio; Frasson, Roberta; Palmieri, Michele; Pazzagli, Mario; De Filippis, Vincenzo; Garaci, Enrico; Ziche, Marina

    2011-12-01

    Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

  20. The endless tale of non-homologous end-joining.

    Science.gov (United States)

    Weterings, Eric; Chen, David J

    2008-01-01

    DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.

  1. Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition.

    Science.gov (United States)

    Tessé, Sophie; Storlazzi, Aurora; Kleckner, Nancy; Gargano, Silvana; Zickler, Denise

    2003-10-28

    Ski8p is implicated in degradation of non-poly(A) and double-stranded RNA, and in meiotic DNA recombination. We have identified the Sordaria macrospora SKI8 gene. Ski8p is cytoplasmically localized in all vegetative and sexual cycle cells, and is nuclear localized, specifically in early-mid-meiotic prophase, in temporal correlation with Spo11p, the meiotic double-strand break (DSB) transesterase. Localizations of Ski8p and Spo11p are mutually interdependent. ski8 mutants exhibit defects in vegetative growth, entry into the sexual program, and sporulation. Diverse meiotic defects, also seen in spo11 mutants, are diagnostic of DSB absence, and they are restored by exogenous DSBs. These results suggest that Ski8p promotes meiotic DSB formation by acting directly within meiotic prophase chromosomes. Mutant phenotypes also divide meiotic homolog juxtaposition into three successive, mechanistically distinct steps; recognition, presynaptic alignment, and synapsis, which are distinguished by their differential dependence on DSBs.

  2. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    International Nuclear Information System (INIS)

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory

    2007-01-01

    Frog virus 3 (FV3) is a large DNA virus that encodes ∼ 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIα). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIα triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins

  3. In Silico Characterization and Structural Modeling of Dermacentor andersoni p36 Immunosuppressive Protein

    Directory of Open Access Journals (Sweden)

    Martin Omulindi Oyugi

    2018-01-01

    Full Text Available Ticks cause approximately $17–19 billion economic losses to the livestock industry globally. Development of recombinant antitick vaccine is greatly hindered by insufficient knowledge and understanding of proteins expressed by ticks. Ticks secrete immunosuppressant proteins that modulate the host’s immune system during blood feeding; these molecules could be a target for antivector vaccine development. Recombinant p36, a 36 kDa immunosuppressor from the saliva of female Dermacentor andersoni, suppresses T-lymphocytes proliferation in vitro. To identify potential unique structural and dynamic properties responsible for the immunosuppressive function of p36 proteins, this study utilized bioinformatic tool to characterize and model structure of D. andersoni p36 protein. Evaluation of p36 protein family as suitable vaccine antigens predicted a p36 homolog in Rhipicephalus appendiculatus, the tick vector of East Coast fever, with an antigenicity score of 0.7701 that compares well with that of Bm86 (0.7681, the protein antigen that constitute commercial tick vaccine Tickgard™. Ab initio modeling of the D. andersoni p36 protein yielded a 3D structure that predicted conserved antigenic region, which has potential of binding immunomodulating ligands including glycerol and lactose, found located within exposed loop, suggesting a likely role in immunosuppressive function of tick p36 proteins. Laboratory confirmation of these preliminary results is necessary in future studies.

  4. Protein structure analysis using the resonant recognition model and wavelet transforms

    International Nuclear Information System (INIS)

    Fang, Q.; Cosic, I.

    1998-01-01

    An approach based on the resonant recognition model and the discrete wavelet transform is introduced here for characterising proteins' biological function. The protein sequence is converted into a numerical series by assigning the electron-ion interaction potential to each amino acid from N-terminal to C-terminal. A set of peaks is found after performing a wavelet transform onto a numerical series representing a group of homologous proteins. These peaks are related to protein structural and functional properties and named characteristic vector of that protein group. Further more, the amino acids contributing mostly to a protein's biological functions, the so-called 'hot spots' amino acids, are predicted by the continuous wavelet transform. It is found that the hot spots are clustered around the protein's cleft structure. The wavelets approach provides a novel methods for amino acid sequence analysis as well as an expansion for the newly established macromolecular interaction model: the resonant recognition model. Copyright (1998) Australasian Physical and Engineering Sciences in Medicine

  5. Fast loop modeling for protein structures

    Science.gov (United States)

    Zhang, Jiong; Nguyen, Son; Shang, Yi; Xu, Dong; Kosztin, Ioan

    2015-03-01

    X-ray crystallography is the main method for determining 3D protein structures. In many cases, however, flexible loop regions of proteins cannot be resolved by this approach. This leads to incomplete structures in the protein data bank, preventing further computational study and analysis of these proteins. For instance, all-atom molecular dynamics (MD) simulation studies of structure-function relationship require complete protein structures. To address this shortcoming, we have developed and implemented an efficient computational method for building missing protein loops. The method is database driven and uses deep learning and multi-dimensional scaling algorithms. We have implemented the method as a simple stand-alone program, which can also be used as a plugin in existing molecular modeling software, e.g., VMD. The quality and stability of the generated structures are assessed and tested via energy scoring functions and by equilibrium MD simulations. The proposed method can also be used in template-based protein structure prediction. Work supported by the National Institutes of Health [R01 GM100701]. Computer time was provided by the University of Missouri Bioinformatics Consortium.

  6. Molecular modelling of the Norrie disease protein predicts a cystine knot growth factor tertiary structure.

    Science.gov (United States)

    Meitinger, T; Meindl, A; Bork, P; Rost, B; Sander, C; Haasemann, M; Murken, J

    1993-12-01

    The X-lined gene for Norrie disease, which is characterized by blindness, deafness and mental retardation has been cloned recently. This gene has been thought to code for a putative extracellular factor; its predicted amino acid sequence is homologous to the C-terminal domain of diverse extracellular proteins. Sequence pattern searches and three-dimensional modelling now suggest that the Norrie disease protein (NDP) has a tertiary structure similar to that of transforming growth factor beta (TGF beta). Our model identifies NDP as a member of an emerging family of growth factors containing a cystine knot motif, with direct implications for the physiological role of NDP. The model also sheds light on sequence related domains such as the C-terminal domain of mucins and of von Willebrand factor.

  7. Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the arginine deiminase system of Streptococcus pyogenes

    NARCIS (Netherlands)

    Winterhoff, N.; Goethe, R.; Gruening, P.; Rohde, M.; Kalisz, H.; Smith, H.E.; Valentin-Weigand, P.

    2002-01-01

    The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42°C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42°C

  8. Models of crk adaptor proteins in cancer.

    Science.gov (United States)

    Bell, Emily S; Park, Morag

    2012-05-01

    The Crk family of adaptor proteins (CrkI, CrkII, and CrkL), originally discovered as the oncogene fusion product, v-Crk, of the CT10 chicken retrovirus, lacks catalytic activity but engages with multiple signaling pathways through their SH2 and SH3 domains. Crk proteins link upstream tyrosine kinase and integrin-dependent signals to downstream effectors, acting as adaptors in diverse signaling pathways and cellular processes. Crk proteins are now recognized to play a role in the malignancy of many human cancers, stimulating renewed interest in their mechanism of action in cancer progression. The contribution of Crk signaling to malignancy has been predominantly studied in fibroblasts and in hematopoietic models and more recently in epithelial models. A mechanistic understanding of Crk proteins in cancer progression in vivo is still poorly understood in part due to the highly pleiotropic nature of Crk signaling. Recent advances in the structural organization of Crk domains, new roles in kinase regulation, and increased knowledge of the mechanisms and frequency of Crk overexpression in human cancers have provided an incentive for further study in in vivo models. An understanding of the mechanisms through which Crk proteins act as oncogenic drivers could have important implications in therapeutic targeting.

  9. The Phyre2 web portal for protein modeling, prediction and analysis.

    Science.gov (United States)

    Kelley, Lawrence A; Mezulis, Stefans; Yates, Christopher M; Wass, Mark N; Sternberg, Michael J E

    2015-06-01

    Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.

  10. Validation-driven protein-structure improvement

    NARCIS (Netherlands)

    Touw, W.G.

    2016-01-01

    High-quality protein structure models are essential for many Life Science applications, such as protein engineering, molecular dynamics, drug design, and homology modelling. The WHAT_CHECK model validation project and the PDB_REDO model optimisation project have shown that many structure models in

  11. Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report

    Directory of Open Access Journals (Sweden)

    Heffer Alison

    2011-03-01

    Full Text Available Abstract Background Tremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly. Results Here we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR and gene-specific amplified fragment length polymorphism (AFLP. It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution. Conclusions RIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life.

  12. Baculovirus replication: characterization of DNA and proteins synthesized by a nuclear polyhedrosis virus of Lymantria dispar, the gypsy moth, in a homologous cell line

    International Nuclear Information System (INIS)

    McClintock, J.T.

    1985-01-01

    A multiple-embedded nuclear polyhedrosis virus (NPV) of the gypsy moth, Lymantria dispar (LdMNPV), is used for biological control. However, LdMNPV has low natural virulence and a long infection cycle in relation to other NPVs. Therefore, the replicative cycle of LdMNPV was investigated using a homologous cell line, IPLB-LD-652Y. Based on analyses of virus growth curves LdMNPV nonoccluded virus and polyhedral inclusion bodies appeared approximately 20 and 50 hr postinfection (p.i.), respectively. LdMNPV polypeptides, identified by autoradiography of [ 35 S]-methionine labeled fractions in SDS-PAGE, were synthesized in sequential phases: (1) an early α phase of replication (4 polypeptides from 4 to 12 hr p.i.), (2) an intermediate β phase (20 polypeptides from 12 to 24 hr p.i.), and a late γ phase (4 polypeptides from 24 to 28 hr p.i.). In infected cells at least four polypeptides were post-translational cleaved and/or modified. Pulse-labeling with [ 3 H]-mannose, [ 3 H]-N-acetyl-glucosamine or [ 32 P]-monosodium phosphate revealed several viral polypeptides which were glycosylated and/or phosphorylated. DNA:DNA dot hybridization experiments suggested that LdMNPV DNA synthesis was initiated between 12 to 16 hr p.i., increasing significantly thereafter

  13. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

    Directory of Open Access Journals (Sweden)

    Morrical Scott W

    2010-12-01

    Full Text Available Abstract Homologous recombination (HR, a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR. T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

  14. Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15.

    OpenAIRE

    Heckenlively, J R; Chang, B; Erway, L C; Peng, C; Hawes, N L; Hageman, G S; Roderick, T H

    1995-01-01

    Usher syndrome is a group of diseases with autosomal recessive inheritance, congenital hearing loss, and the development of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and visual field loss over several decades. The causes of Usher syndrome are unknown and no animal models have been available for study. Four human gene sites have been reported, suggesting at least four separate forms of Usher syndrome. We report a mouse model of type I Usher syndr...

  15. Hydration dynamics near a model protein surface

    International Nuclear Information System (INIS)

    Russo, Daniela; Hura, Greg; Head-Gordon, Teresa

    2003-01-01

    The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering and molecular dynamics simulation for both the completely deuterated and completely hydrogenated leucine monomer. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational water dynamics are analyzed in a first approximation with a jump diffusion model. At the highest solute concentrations, the hydration water dynamics is significantly suppressed and characterized by a long residential time and a slow diffusion coefficient. The analysis of the more dilute concentration solutions takes into account the results of the 2.0M solution as a model of the first hydration shell. Subtracting the first hydration layer based on the 2.0M spectra, the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis shows spatially heterogeneous dynamics at high concentration that becomes homogeneous at more dilute concentrations. We discuss the hydration dynamics results of this model protein system in the context of glassy systems, protein function, and protein-protein interfaces

  16. eMatchSite: sequence order-independent structure alignments of ligand binding pockets in protein models.

    Directory of Open Access Journals (Sweden)

    Michal Brylinski

    2014-09-01

    Full Text Available Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4-9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite.

  17. Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G; Choudhary, Neelima; Routh, Patty A; Cholon, Deborah M; Hobbs, Marcia M; Almond, Glen W; Orndorff, Paul E; Elkins, Christopher

    2011-08-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

  18. Passive Immunization with a Polyclonal Antiserum to the Hemoglobin Receptor of Haemophilus ducreyi Confers Protection against a Homologous Challenge in the Experimental Swine Model of Chancroid▿

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G.; Choudhary, Neelima; Routh, Patty A.; Cholon, Deborah M.; Hobbs, Marcia M.; Almond, Glen W.; Orndorff, Paul E.; Elkins, Christopher

    2011-01-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme. PMID:21646451

  19. Predicting Protein Secondary Structure with Markov Models

    DEFF Research Database (Denmark)

    Fischer, Paul; Larsen, Simon; Thomsen, Claus

    2004-01-01

    we are considering here, is to predict the secondary structure from the primary one. To this end we train a Markov model on training data and then use it to classify parts of unknown protein sequences as sheets, helices or coils. We show how to exploit the directional information contained...... in the Markov model for this task. Classifications that are purely based on statistical models might not always be biologically meaningful. We present combinatorial methods to incorporate biological background knowledge to enhance the prediction performance....

  20. Examination of the requirement for ucp-4, a putative homolog of mammalian uncoupling proteins, for stress tolerance and longevity in C. elegans.

    Science.gov (United States)

    Iser, Wendy B; Kim, Daemyung; Bachman, Eric; Wolkow, Catherine

    2005-10-01

    Reactive oxygen species (ROS) are generated by mitochondrial respiration and can react with and damage cellular components. According to the free radical theory of aging, oxidative damage from mitochondrial ROS is a major cause of cellular decline during aging. Mitochondrial uncoupling proteins (UCPs) uncouple ATP production from electron transport and can be stimulated by free radicals, suggesting UCPs may perform a cytoprotective function. The nematode, Caenorhabditis elegans, contains one UCP-like protein, encoded by the ucp-4 gene. We have investigated the genetic requirement for ucp-4 in normal aging and stress resistance. Consistent with the hypothesis that ucp-4 encodes a putative uncoupling protein, animals lacking ucp-4 function contained elevated ATP levels. However, the absence of ucp-4 function did not affect adult lifespan or survival in the presence of thermal or oxidative stress. Together, these results demonstrate that ucp-4 is a negative regulator of ATP production in C. elegans, but is not required for normal lifespan.

  1. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog

    International Nuclear Information System (INIS)

    Buss, J.E.; Sefton, B.M.

    1985-01-01

    The lipid bound to p60/sub src/, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [ 3 H]myristic acid or [ 3 H]palmitic acid. Incorporation of [ 3 H]myristic acid was noticeably greater than incorporation of [ 3 H]palmitic acid. All of the [ 3 H]myristic acid-derived label in p60/sub src/ was present as myristic acid. In contrast, none of the radioactivity derived from [ 3 H]palmitic acid was recovered as palmitic acid. Instead, all 3 H incorporated into p60/sub src/ from [ 3 H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-/sub src/ also contains myristic acid. By comparison of the extent of myristylation of p60v-/sub src/ with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, the authors estimate that greater than 80% of the molecules of p60v-/sub src/ contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-/sub src/ contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [ 3 H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [ 3 H]myristic acid-labeled p60/sub src/ in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60/sub src/ present in cells

  2. Protein Folding: Search for Basic Physical Models

    Directory of Open Access Journals (Sweden)

    Ivan Y. Torshin

    2003-01-01

    Full Text Available How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context of in vivo protein folding (which has been studied only for a few proteins, the roles of the fundamental physical forces in the in vitro folding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces. Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

  3. The Th1 immune response to Plasmodium falciparum circumsporozoite protein is boosted by adenovirus vectors 35 and 26 with a homologous insert

    NARCIS (Netherlands)

    Radosevic, Katarina; Rodriguez, Ariane; Lemckert, Angelique A. C.; van der Meer, Marjolein; Gillissen, Gert; Warnar, Carolien; von Eyben, Rie; Pau, Maria Grazia; Goudsmit, Jaap

    2010-01-01

    The most advanced malaria vaccine, RTS,S, is comprised of an adjuvant portion of the Plasmodium falciparum circumsporozoite (CS) protein fused to and admixed with the hepatitis B virus surface antigen. This vaccine confers short-term protection against malaria infection, with an efficacy of about

  4. p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair.

    NARCIS (Netherlands)

    S. Humbert; H. van Vuuren; Y. Lutz; J.H.J. Hoeijmakers (Jan); J-M. Egly (Jean-Marc); V. Moncollin

    1994-01-01

    textabstractThe human BTF2 (TFIIH) transcription factor is a multisubunit protein involved in transcription initiation by RNA polymerase II (B) as well as in DNA repair. In addition to the previously characterized p62 and p89/ERCC3 subunits, we have cloned two other subunits of BTF2, p44 and p34.

  5. Relative contributions of decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 in the protection of melanocytes from homologous complement

    NARCIS (Netherlands)

    Venneker, G. T.; Vodegel, R. M.; Okada, N.; Westerhof, W.; Bos, J. D.; Asghar, S. S.

    1998-01-01

    Complement regulatory molecules, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, protect body cells from autologous complement. They have wide tissue distribution but nothing is known about the expression of these molecules on human melanocytes. Since melanocytes are lysed

  6. Inhibitory Effect of Berberine on Zeste Homolog 2 (Ezh2 ...

    African Journals Online (AJOL)

    homolog 2 (Ezh2) expressions in KYSE450 human esophageal cancer cells. Methods: ... of the AXL receptor kinase. The results of ... effects of estrogen receptor antagonists on ..... protein EZH2 is involved in progression of prostate cancer.

  7. Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

    Science.gov (United States)

    Zhao, Hongyu; Yao, Zhenyu; Chen, Xiangbin; Wang, Xinquan; Chen, Guo-Qiang

    2017-11-01

    Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaP A h from Aeromonas hydrophila has been solved to date. Based on the structure of PhaP A h , homology models of PhaP A z from Azotobacter sp. FA-8 and PhaP TD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaP A z mutants, including PhaP A z Q38L and PhaP A z Q78L, as well as PhaP TD mutants, including PhaP TD Q38M and PhaP TD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaP A h , can be generalized to facilitate the study of other PhaP members. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  8. Homology modeling and docking analyses of M. leprae Mur ligases reveals the common binding residues for structure based drug designing to eradicate leprosy.

    Science.gov (United States)

    Shanmugam, Anusuya; Natarajan, Jeyakumar

    2012-06-01

    Multi drug resistance capacity for Mycobacterium leprae (MDR-Mle) demands the profound need for developing new anti-leprosy drugs. Since most of the drugs target a single enzyme, mutation in the active site renders the antibiotic ineffective. However, structural and mechanistic information on essential bacterial enzymes in a pathway could lead to the development of antibiotics that targets multiple enzymes. Peptidoglycan is an important component of the cell wall of M. leprae. The biosynthesis of bacterial peptidoglycan represents important targets for the development of new antibacterial drugs. Biosynthesis of peptidoglycan is a multi-step process that involves four key Mur ligase enzymes: MurC (EC:6.3.2.8), MurD (EC:6.3.2.9), MurE (EC:6.3.2.13) and MurF (EC:6.3.2.10). Hence in our work, we modeled the three-dimensional structure of the above Mur ligases using homology modeling method and analyzed its common binding features. The residues playing an important role in the catalytic activity of each of the Mur enzymes were predicted by docking these Mur ligases with their substrates and ATP. The conserved sequence motifs significant for ATP binding were predicted as the probable residues for structure based drug designing. Overall, the study was successful in listing significant and common binding residues of Mur enzymes in peptidoglycan pathway for multi targeted therapy.

  9. Modeling disordered regions in proteins using Rosetta.

    Directory of Open Access Journals (Sweden)

    Ray Yu-Ruei Wang

    Full Text Available Protein structure prediction methods such as Rosetta search for the lowest energy conformation of the polypeptide chain. However, the experimentally observed native state is at a minimum of the free energy, rather than the energy. The neglect of the missing configurational entropy contribution to the free energy can be partially justified by the assumption that the entropies of alternative folded states, while very much less than unfolded states, are not too different from one another, and hence can be to a first approximation neglected when searching for the lowest free energy state. The shortcomings of current structure prediction methods may be due in part to the breakdown of this assumption. Particularly problematic are proteins with significant disordered regions which do not populate single low energy conformations even in the native state. We describe two approaches within the Rosetta structure modeling methodology for treating such regions. The first does not require advance knowledge of the regions likely to be disordered; instead these are identified by minimizing a simple free energy function used previously to model protein folding landscapes and transition states. In this model, residues can be either completely ordered or completely disordered; they are considered disordered if the gain in entropy outweighs the loss of favorable energetic interactions with the rest of the protein chain. The second approach requires identification in advance of the disordered regions either from sequence alone using for example the DISOPRED server or from experimental data such as NMR chemical shifts. During Rosetta structure prediction calculations the disordered regions make only unfavorable repulsive contributions to the total energy. We find that the second approach has greater practical utility and illustrate this with examples from de novo structure prediction, NMR structure calculation, and comparative modeling.

  10. Comprehensive analysis of interactions between the Src-associated protein in mitosis of 68 kDa and the human Src-homology 3 proteome.

    Directory of Open Access Journals (Sweden)

    Benedikt Asbach

    Full Text Available The protein Sam68 is involved in many cellular processes such as cell-cycle regulation, RNA metabolism, or signal transduction. Sam68 comprises a central RNA-binding domain flanked by unstructured tails containing docking sites for signalling proteins including seven proline-rich sequences (denoted P0 to P6 as potential SH3-domain binding motifs. To comprehensively assess Sam68-SH3-interactions, we applied a phage-display screening of a library containing all approx. 300 human SH3 domains. Thereby we identified five new (from intersectin 2, the osteoclast stimulating factor OSF, nephrocystin, sorting nexin 9, and CIN85 and seven already known high-confidence Sam68-ligands (mainly from the Src-kinase family, as well as several lower-affinity binders. Interaction of the high-affinity Sam68-binders was confirmed in independent assays in vitro (phage-ELISA, GST-pull-down and in vivo (FACS-based FRET-analysis with CFP- and YFP-tagged proteins. Fine-mapping analyses with peptides established P0, P3, P4, and P5 as exclusive docking-sites for SH3 domains, which showed varying preferences for these motifs. Mutational analyses identified individual residues within the proline-rich motifs being crucial for the interactions. Based on these data, we generated a Sam68-mutant incapable of interacting with SH3 domains any more, as subsequently demonstrated by FRET-analyses. In conclusion, we present a thorough characterization of Sam68's interplay with the SH3 proteome. The observed interaction between Sam68 and OSF complements the known Sam68-Src and OSF-Src interactions. Thus, we propose, that Sam68 functions as a classical scaffold protein in this context, assembling components of an osteoclast-specific signalling pathway.

  11. Variation in a surface-exposed region of the Mycoplasma pneumoniae P40 protein as a consequence of homologous DNA recombination between RepMP5 elements.

    Science.gov (United States)

    Spuesens, Emiel B M; van de Kreeke, Nick; Estevão, Silvia; Hoogenboezem, Theo; Sluijter, Marcel; Hartwig, Nico G; van Rossum, Annemarie M C; Vink, Cornelis

    2011-02-01

    Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory tract infections. The first step in infection is adherence of the bacteria to the respiratory epithelium. This step is mediated by a specialized organelle, which contains several proteins (cytadhesins) that have an important function in adherence. Two of these cytadhesins, P40 and P90, represent the proteolytic products from a single 130 kDa protein precursor, which is encoded by the MPN142 gene. Interestingly, MPN142 contains a repetitive DNA element, termed RepMP5, of which homologues are found at seven other loci within the M. pneumoniae genome. It has been hypothesized that these RepMP5 elements, which are similar but not identical in sequence, recombine with their counterpart within MPN142 and thereby provide a source of sequence variation for this gene. As this variation may give rise to amino acid changes within P40 and P90, the recombination between RepMP5 elements may constitute the basis of antigenic variation and, possibly, immune evasion by M. pneumoniae. To investigate the sequence variation of MPN142 in relation to inter-RepMP5 recombination, we determined the sequences of all RepMP5 elements in a collection of 25 strains. The results indicate that: (i) inter-RepMP5 recombination events have occurred in seven of the strains, and (ii) putative RepMP5 recombination events involving MPN142 have induced amino acid changes in a surface-exposed part of the P40 protein in two of the strains. We conclude that recombination between RepMP5 elements is a common phenomenon that may lead to sequence variation of MPN142-encoded proteins.

  12. Protein Simulation Data in the Relational Model.

    Science.gov (United States)

    Simms, Andrew M; Daggett, Valerie

    2012-10-01

    High performance computing is leading to unprecedented volumes of data. Relational databases offer a robust and scalable model for storing and analyzing scientific data. However, these features do not come without a cost-significant design effort is required to build a functional and efficient repository. Modeling protein simulation data in a relational database presents several challenges: the data captured from individual simulations are large, multi-dimensional, and must integrate with both simulation software and external data sites. Here we present the dimensional design and relational implementation of a comprehensive data warehouse for storing and analyzing molecular dynamics simulations using SQL Server.

  13. Modeling of the structure of ribosomal protein L1 from the archaeon Haloarcula marismortui

    Science.gov (United States)

    Nevskaya, N. A.; Kljashtorny, V. G.; Vakhrusheva, A. V.; Garber, M. B.; Nikonov, S. V.

    2017-07-01

    The halophilic archaeon Haloarcula marismortui proliferates in the Dead Sea at extremely high salt concentrations (higher than 3 M). This is the only archaeon, for which the crystal structure of the ribosomal 50S subunit was determined. However, the structure of the functionally important side protuberance containing the abnormally negatively charged protein L1 (HmaL1) was not visualized. Attempts to crystallize HmaL1 in the isolated state or as its complex with RNA using normal salt concentrations (≤500 mM) failed. A theoretical model of HmaL1 was built based on the structural data for homologs of the protein L1 from other organisms, and this model was refined by molecular dynamics methods. Analysis of this model showed that the protein HmaL1 can undergo aggregation due to the presence of a cluster of positive charges unique for proteins L1. This cluster is located at the RNA-protein interface, which interferes with the crystallization of HmaL1 and the binding of the latter to RNA.

  14. G protein- and agonist-bound serotonin 5-HT2A receptor model activated by steered molecular dynamics simulations

    DEFF Research Database (Denmark)

    Ísberg, Vignir; Balle, Thomas; Sander, Tommy

    2011-01-01

    molecular dynamics (MD) simulations. The driving force for the transformation was the addition of several known intermolecular and receptor interhelical hydrogen bonds enforcing the necessary helical and rotameric movements. Subsquent MD simulations without constraints confirmed the stability......A 5-HT(2A) receptor model was constructed by homology modeling based on the ß(2)-adrenergic receptor and the G protein-bound opsin crystal structures. The 5-HT(2A) receptor model was transferred into an active conformation by an agonist ligand and a G(aq) peptide in four subsequent steered...

  15. Homology modeling, molecular dynamics, and virtual screening of NorA efflux pump inhibitors of Staphylococcus aureus.

    Science.gov (United States)

    Bhaskar, Baki Vijaya; Babu, Tirumalasetty Muni Chandra; Reddy, Netala Vasudeva; Rajendra, Wudayagiri

    2016-01-01

    Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds.

  16. Persistent homology and string vacua

    Energy Technology Data Exchange (ETDEWEB)

    Cirafici, Michele [Center for Mathematical Analysis, Geometry and Dynamical Systems,Instituto Superior Técnico, Universidade de Lisboa,Av. Rovisco Pais, 1049-001 Lisboa (Portugal); Institut des Hautes Études Scientifiques,Le Bois-Marie, 35 route de Chartres, F-91440 Bures-sur-Yvette (France)

    2016-03-08

    We use methods from topological data analysis to study the topological features of certain distributions of string vacua. Topological data analysis is a multi-scale approach used to analyze the topological features of a dataset by identifying which homological characteristics persist over a long range of scales. We apply these techniques in several contexts. We analyze N=2 vacua by focusing on certain distributions of Calabi-Yau varieties and Landau-Ginzburg models. We then turn to flux compactifications and discuss how we can use topological data analysis to extract physical information. Finally we apply these techniques to certain phenomenologically realistic heterotic models. We discuss the possibility of characterizing string vacua using the topological properties of their distributions.

  17. Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis

    KAUST Repository

    Witz, Sandra

    2014-03-12

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.

  18. Expression, refolding and spectroscopic characterization of fibronectin type III (FnIII)-homology domains derived from human fibronectin leucine rich transmembrane protein (FLRT)-1,-2, and-3

    DEFF Research Database (Denmark)

    Yang, Lila; Falkesgaard, Maria Hansen; Thulstrup, Peter Waaben

    2017-01-01

    various species have been determined, the expression and purification of recombinant FLRT FnIII domains, important steps for further structural and functional characterizations of the proteins, have not yet been described. Here we present a protocol for expressing recombinant FLRT-FnIII domains...... that a strand-strand cystine bridge has significant effect on the stability of the FLRT FnIII fold. We further show by Surface Plasmon Resonance that all three FnIII domains bind to FGFR1, and roughly estimate a Kd for each domain, all Kds being in the µM range....

  19. Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles

    DEFF Research Database (Denmark)

    Hummel, R; Nørgaard, P; Andreasen, P H

    1992-01-01

    The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically...... of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance...

  20. Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains♦

    Science.gov (United States)

    Tanaka, Hiroaki; Akagi, Ken-ichi; Oneyama, Chitose; Tanaka, Masakazu; Sasaki, Yuichi; Kanou, Takashi; Lee, Young-Ho; Yokogawa, Daisuke; Dobenecker, Marc-Werner; Nakagawa, Atsushi; Okada, Masato; Ikegami, Takahisa

    2013-01-01

    Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. PMID:23548896

  1. Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

    International Nuclear Information System (INIS)

    Rana, Ritu; Jagadish, Nirmala; Garg, Manoj; Mishra, Deepshikha; Dahiya, Neetu; Chaurasiya, Dipak; Suri, Anil

    2006-01-01

    Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology

  2. Markov state models of protein misfolding

    Science.gov (United States)

    Sirur, Anshul; De Sancho, David; Best, Robert B.

    2016-02-01

    Markov state models (MSMs) are an extremely useful tool for understanding the conformational dynamics of macromolecules and for analyzing MD simulations in a quantitative fashion. They have been extensively used for peptide and protein folding, for small molecule binding, and for the study of native ensemble dynamics. Here, we adapt the MSM methodology to gain insight into the dynamics of misfolded states. To overcome possible flaws in root-mean-square deviation (RMSD)-based metrics, we introduce a novel discretization approach, based on coarse-grained contact maps. In addition, we extend the MSM methodology to include "sink" states in order to account for the irreversibility (on simulation time scales) of processes like protein misfolding. We apply this method to analyze the mechanism of misfolding of tandem repeats of titin domains, and how it is influenced by confinement in a chaperonin-like cavity.

  3. Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins

    DEFF Research Database (Denmark)

    Rognan, D; Lauemoller, S L; Holm, A

    1999-01-01

    A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein...... coordinates of the MHC-bound peptide have first been determined with an accuracy of about 1-1.5 A. Furthermore, it may be easily recalibrated for any protein-ligand complex.......) and of a series of 16 peptides to H-2K(k). Predictions were more accurate for HLA-A2-binding peptides as the training set had been built from experimentally determined structures. The average error in predicting the binding free energy of the test peptides was 3.1 kJ/mol. For the homology model-derived equation...

  4. SANSparallel: interactive homology search against Uniprot.

    Science.gov (United States)

    Somervuo, Panu; Holm, Liisa

    2015-07-01

    Proteins evolve by mutations and natural selection. The network of sequence similarities is a rich source for mining homologous relationships that inform on protein structure and function. There are many servers available to browse the network of homology relationships but one has to wait up to a minute for results. The SANSparallel webserver provides protein sequence database searches with immediate response and professional alignment visualization by third-party software. The output is a list, pairwise alignment or stacked alignment of sequence-similar proteins from Uniprot, UniRef90/50, Swissprot or Protein Data Bank. The stacked alignments are viewed in Jalview or as sequence logos. The database search uses the suffix array neighborhood search (SANS) method, which has been re-implemented as a client-server, improved and parallelized. The method is extremely fast and as sensitive as BLAST above 50% sequence identity. Benchmarks show that the method is highly competitive compared to previously published fast database search programs: UBLAST, DIAMOND, LAST, LAMBDA, RAPSEARCH2 and BLAT. The web server can be accessed interactively or programmatically at http://ekhidna2.biocenter.helsinki.fi/cgi-bin/sans/sans.cgi. It can be used to make protein functional annotation pipelines more efficient, and it is useful in interactive exploration of the detailed evidence supporting the annotation of particular proteins of interest. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. [Expression of erythroblastic leukemia viral oncogene homolog 3 (ErbB-3) binding protein-1, matrix metalloproteinases, eplthelial cadherin in adenoid cystic carcinoma and correlation analysis].

    Science.gov (United States)

    Sun, Jian; Yu, You-cheng; Luo, Yi-xi; Tian, Zhen

    2012-12-01

    To investigate the expression of ErbB-3 binding protein-1 (EBP-1), matrix metalloproteinase 9 (MMP-9) and E-cadherin (E-cad) in adenoid cystic carcinoma and their correlation. Immunohistochemistry(PV6000 method) was used to detect EBP-1, MMP-9 and E-cad expression in 66 cases of adenoid cystic carcinoma tissues and matched para-cancerous normal tissues. In this study all cases were successfully followed up. The positive expression rate of EBP-1 in adenoid cystic carcinoma tissues was 85%. EBP-1 expression was significantly correlated to pathological pattern and clinical stage (P correlation between EBP-1 and E-cad expression, and positive correlation between EBP-1 and MMP-9. EBP-1 and its correlation with MMP-9 and E-cad may be used as useful indicators for clinical assessment of tumor biological behavior and prognosis in patients with adenoid cystic carcinoma.

  6. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Karin [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden); Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Grawé, Jan [Department of Genetics and Pathology, Uppsala University, Uppsala 75185 (Sweden); McKinney-Freeman, Shannon L. [Department of Hematology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Daley, George Q. [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Welsh, Michael, E-mail: michael.welsh@mcb.uu.se [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden)

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via

  7. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    International Nuclear Information System (INIS)

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-01-01

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  8. Comparative sequence and structural analyses of G-protein-coupled receptor crystal structures and implications for molecular models.

    Directory of Open Access Journals (Sweden)

    Catherine L Worth

    Full Text Available BACKGROUND: Up until recently the only available experimental (high resolution structure of a G-protein-coupled receptor (GPCR was that of bovine rhodopsin. In the past few years the determination of GPCR structures has accelerated with three new receptors, as well as squid rhodopsin, being successfully crystallized. All share a common molecular architecture of seven transmembrane helices and can therefore serve as templates for building molecular models of homologous GPCRs. However, despite the common general architecture of these structures key differences do exist between them. The choice of which experimental GPCR structure(s to use for building a comparative model of a particular GPCR is unclear and without detailed structural and sequence analyses, could be arbitrary. The aim of this study is therefore to perform a systematic and detailed analysis of sequence-structure relationships of known GPCR structures. METHODOLOGY: We analyzed in detail conserved and unique sequence motifs and structural features in experimentally-determined GPCR structures. Deeper insight into specific and important structural features of GPCRs as well as valuable information for template selection has been gained. Using key features a workflow has been formulated for identifying the most appropriate template(s for building homology models of GPCRs of unknown structure. This workflow was applied to a set of 14 human family A GPCRs suggesting for each the most appropriate template(s for building a comparative molecular model. CONCLUSIONS: The available crystal structures represent only a subset of all possible structural variation in family A GPCRs. Some GPCRs have structural features that are distributed over different crystal structures or which are not present in the templates suggesting that homology models should be built using multiple templates. This study provides a systematic analysis of GPCR crystal structures and a consistent method for identifying

  9. Comparative sequence and structural analyses of G-protein-coupled receptor crystal structures and implications for molecular models.

    Science.gov (United States)

    Worth, Catherine L; Kleinau, Gunnar; Krause, Gerd

    2009-09-16

    Up until recently the only available experimental (high resolution) structure of a G-protein-coupled receptor (GPCR) was that of bovine rhodopsin. In the past few years the determination of GPCR structures has accelerated with three new receptors, as well as squid rhodopsin, being successfully crystallized. All share a common molecular architecture of seven transmembrane helices and can therefore serve as templates for building molecular models of homologous GPCRs. However, despite the common general architecture of these structures key differences do exist between them. The choice of which experimental GPCR structure(s) to use for building a comparative model of a particular GPCR is unclear and without detailed structural and sequence analyses, could be arbitrary. The aim of this study is therefore to perform a systematic and detailed analysis of sequence-structure relationships of known GPCR structures. We analyzed in detail conserved and unique sequence motifs and structural features in experimentally-determined GPCR structures. Deeper insight into specific and important structural features of GPCRs as well as valuable information for template selection has been gained. Using key features a workflow has been formulated for identifying the most appropriate template(s) for building homology models of GPCRs of unknown structure. This workflow was applied to a set of 14 human family A GPCRs suggesting for each the most appropriate template(s) for building a comparative molecular model. The available crystal structures represent only a subset of all possible structural variation in family A GPCRs. Some GPCRs have structural features that are distributed over different crystal structures or which are not present in the templates suggesting that homology models should be built using multiple templates. This study provides a systematic analysis of GPCR crystal structures and a consistent method for identifying suitable templates for GPCR homology modelling that will

  10. The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

    Directory of Open Access Journals (Sweden)

    Tam Michael WC

    2010-03-01

    Full Text Available Abstract Background Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. Results The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1 RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. Conclusions The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to

  11. Induction of homologous recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Simon, J R; Moore, P D

    1988-09-01

    We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

  12. Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates.

    Science.gov (United States)

    Aouacheria, Abdel; Geourjon, Christophe; Aghajari, Nushin; Navratil, Vincent; Deléage, Gilbert; Lethias, Claire; Exposito, Jean-Yves

    2006-12-01

    Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.

  13. The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7.

    Science.gov (United States)

    Gabus, C; Ficheux, D; Rau, M; Keith, G; Sandmeyer, S; Darlix, J L

    1998-08-17

    Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.

  14. Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450.

    Science.gov (United States)

    Ewen, Kerstin Maria; Hannemann, Frank; Khatri, Yogan; Perlova, Olena; Kappl, Reinhard; Krug, Daniel; Hüttermann, Jürgen; Müller, Rolf; Bernhardt, Rita

    2009-10-16

    Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.

  15. Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

    Directory of Open Access Journals (Sweden)

    Emmanuel A Asante

    Full Text Available Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP gene (PRNP and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP(Sc, pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP ((CtmPrP. Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP(Sc was demonstrated in the brains of recipient transgenic mice. This PrP(Sc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of (CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

  16. Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

    Science.gov (United States)

    Asante, Emmanuel A; Linehan, Jacqueline M; Smidak, Michelle; Tomlinson, Andrew; Grimshaw, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Powell, Caroline; Brandner, Sebastian; Wadsworth, Jonathan D F; Collinge, John

    2013-01-01

    Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP(Sc)), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP ((Ctm)PrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP(Sc) was demonstrated in the brains of recipient transgenic mice. This PrP(Sc) rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of (Ctm)PrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

  17. Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

    Energy Technology Data Exchange (ETDEWEB)

    Kurashima-Ito, Kaori [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Ikeya, Teppei [National Institute of Advanced Industrial Science and Technology (AIST), (Japan); Senbongi, Hiroshi [Mitochondrial Diseases Group, MRC Dunn Human NutritionUnit (United Kingdom); Tochio, Hidehito [International Graduate School of Arts and Sciences, Supramolecular Biology, Yokohama City University, Molecular Biophysics Laboratory (Japan); Mikawa, Tsutomu [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Shibata, Takehiko [RIKEN, Shibata Distinguished Senior Scientist Laboratory (Japan); Ito, Yutaka [RIKEN, Cellular and Molecular Biology Laboratory (Japan)], E-mail: ito-yutaka@center.tmu.ac.jp

    2006-05-15

    Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired {sup 13}C and {sup 15}N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and {alpha}/{beta}-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle.

  18. Ultrafast Proton Shuttling in Psammocora Cyan Fluorescent Protein

    NARCIS (Netherlands)

    Kennis, J.T.M.; van Stokkum, I.H.M.; Peterson, D.S.; Pandit, A.; Wachter, R.M.

    2013-01-01

    Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy

  19. Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

    Science.gov (United States)

    Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

    2009-04-01

    Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

  20. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    Directory of Open Access Journals (Sweden)

    Sonia Gullón

    Full Text Available Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase and a Tat-dependent model protein (agarase in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  1. Statistical alignment: computational properties, homology testing and goodness-of-fit

    DEFF Research Database (Denmark)

    Hein, J; Wiuf, Carsten; Møller, Martin

    2000-01-01

    The model of insertions and deletions in biological sequences, first formulated by Thorne, Kishino, and Felsenstein in 1991 (the TKF91 model), provides a basis for performing alignment within a statistical framework. Here we investigate this model.Firstly, we show how to accelerate the statistical...... alignment algorithms several orders of magnitude. The main innovations are to confine likelihood calculations to a band close to the similarity based alignment, to get good initial guesses of the evolutionary parameters and to apply an efficient numerical optimisation algorithm for finding the maximum...... analysis.Secondly, we propose a new homology test based on this model, where homology means that an ancestor to a sequence pair can be found finitely far back in time. This test has statistical advantages relative to the traditional shuffle test for proteins.Finally, we describe a goodness-of-fit test...

  2. Using structure to explore the sequence alignment space of remote homologs.

    Directory of Open Access Journals (Sweden)

    Andrew Kuziemko

    2011-10-01

    Full Text Available Protein structure modeling by homology requires an accurate sequence alignment between the query protein and its structural template. However, sequence alignment methods based on dynamic programming (DP are typically unable to generate accurate alignments for remote sequence homologs, thus limiting the applicability of modeling methods. A central problem is that the alignment that is "optimal" in terms of the DP score does not necessarily correspond to the alignment that produces the most accurate structural model. That is, the correct alignment based on structural superposition will generally have a lower score than the optimal alignment obtained from sequence. Variations of the DP algorithm have been developed that generate alternative alignments that are "suboptimal" in terms of the DP score, but these still encounter difficulties in detecting the correct structural alignment. We present here a new alternative sequence alignment method that relies heavily on the structure of the template. By initially aligning the query sequence to individual fragments in secondary structure elements and combining high-scoring fragments that pass basic tests for "modelability", we can generate accurate alignments within a small ensemble. Our results suggest that the set of sequences that can currently be modeled by homology can be greatly extended.

  3. Using structure to explore the sequence alignment space of remote homologs.

    Science.gov (United States)

    Kuziemko, Andrew; Honig, Barry; Petrey, Donald

    2011-10-01

    Protein structure modeling by homology requires an accurate sequence alignment between the query protein and its structural template. However, sequence alignment methods based on dynamic programming (DP) are typically unable to generate accurate alignments for remote sequence homologs, thus limiting the applicability of modeling methods. A central problem is that the alignment that is "optimal" in terms of the DP score does not necessarily correspond to the alignment that produces the most accurate structural model. That is, the correct alignment based on structural superposition will generally have a lower score than the optimal alignment obtained from sequence. Variations of the DP algorithm have been developed that generate alternative alignments that are "suboptimal" in terms of the DP score, but these still encounter difficulties in detecting the correct structural alignment. We present here a new alternative sequence alignment method that relies heavily on the structure of the template. By initially aligning the query sequence to individual fragments in secondary structure elements and combining high-scoring fragments that pass basic tests for "modelability", we can generate accurate alignments within a small ensemble. Our results suggest that the set of sequences that can currently be modeled by homology can be greatly extended.

  4. Identification of Oxa1 Homologs Operating in the Eukaryotic Endoplasmic Reticulum

    Directory of Open Access Journals (Sweden)

    S. Andrei Anghel

    2017-12-01

    Full Text Available Members of the evolutionarily conserved Oxa1/Alb3/YidC family mediate membrane protein biogenesis at the mitochondrial inner membrane, chloroplast thylakoid membrane, and bacterial plasma membrane, respectively. Despite their broad phylogenetic distribution, no Oxa1/Alb3/YidC homologs are known to operate in eukaryotic cells outside the endosymbiotic organelles. Here, we present bioinformatic evidence that the tail-anchored protein insertion factor WRB/Get1, the “endoplasmic reticulum (ER membrane complex” subunit EMC3, and TMCO1 are ER-resident homologs of the Oxa1/Alb3/YidC family. Topology mapping and co-evolution-based modeling demonstrate that Get1, EMC3, and TMCO1 share a conserved Oxa1-like architecture. Biochemical analysis of human TMCO1, the only homolog not previously linked to membrane protein biogenesis, shows that it associates with the Sec translocon and ribosomes. These findings suggest a specific biochemical function for TMCO1 and define a superfamily of proteins—the “Oxa1 superfamily”—whose shared function is to facilitate membrane protein biogenesis.

  5. PROCOV: maximum likelihood estimation of protein phylogeny under covarion models and site-specific covarion pattern analysis

    Directory of Open Access Journals (Sweden)

    Wang Huai-Chun

    2009-09-01

    Full Text Available Abstract Background The covarion hypothesis of molecular evolution holds that selective pressures on a given amino acid or nucleotide site are dependent on the identity of other sites in the molecule that change throughout time, resulting in changes of evolutionary rates of sites along the branches of a phylogenetic tree. At the sequence level, covarion-like evolution at a site manifests as conservation of nucleotide or amino acid states among some homologs where the states are not conserved in other homologs (or groups of homologs. Covarion-like evolution has been shown to relate to changes in functions at sites in different clades, and, if ignored, can adversely affect the accuracy of phylogenetic inference. Results PROCOV (protein covarion analysis is a software tool that implements a number of previously proposed covarion models of protein evolution for phylogenetic inference in a maximum likelihood framework. Several algorithmic and implementation improvements in this tool over previous versions make computationally expensive tree searches with covarion models more efficient and analyses of large phylogenomic data sets tractable. PROCOV can be used to identify covarion sites by comparing the site likelihoods under the covarion process to the corresponding site likelihoods under a rates-across-sites (RAS process. Those sites with the greatest log-likelihood difference between a 'covarion' and an RAS process were found to be of functional or structural significance in a dataset of bacterial and eukaryotic elongation factors. Conclusion Covarion models implemented in PROCOV may be especially useful for phylogenetic estimation when ancient divergences between sequences have occurred and rates of evolution at sites are likely to have changed over the tree. It can also be used to study lineage-specific functional shifts in protein families that result in changes in the patterns of site variability among subtrees.

  6. Mod two homology and cohomology

    CERN Document Server

    Hausmann, Jean-Claude

    2014-01-01

    Cohomology and homology modulo 2 helps the reader grasp more readily the basics of a major tool in algebraic topology. Compared to a more general approach to (co)homology this refreshing approach has many pedagogical advantages: It leads more quickly to the essentials of the subject, An absence of signs and orientation considerations simplifies the theory, Computations and advanced applications can be presented at an earlier stage, Simple geometrical interpretations of (co)chains. Mod 2 (co)homology was developed in the first quarter of the twentieth century as an alternative to integral homology, before both became particular cases of (co)homology with arbitrary coefficients. The first chapters of this book may serve as a basis for a graduate-level introductory course to (co)homology. Simplicial and singular mod 2 (co)homology are introduced, with their products and Steenrod squares, as well as equivariant cohomology. Classical applications include Brouwer's fixed point theorem, Poincaré duality, Borsuk-Ula...

  7. A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide

    NARCIS (Netherlands)

    Langedijk, J.P.M.; Veelen, van P.A.; Schaaper, W.M.M.; Ru, de A.H.; Meloen, R.H.; Hulst, M.M.

    2002-01-01

    Erns is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. Erns is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine

  8. p53 regulates the repair of DNA double-strand breaks by both homologous and non-homologous recombination

    International Nuclear Information System (INIS)

    Willers, H.; Powell, S.N.; Dahm-Daphi, J.

    2003-01-01

    Full text: p53 is known to suppress spontaneous homologous recombination (HR), while its role in non-homologous recombination (NHR) remains to be clarified. Here, we sought to determine the influence of p53 on the repair of chromosomal double-strand breaks (DSBs) by HR or NHR using specially designed recombination substrates that integrate into the genome. Isogenic mouse fibroblast pairs with or without expression of exogenous p53 protein were utilized. A reporter plasmid carrying a mutated XGPRT gene was chromosomally integrated and DSBs were generated within the plasmid by the I-SceI endonuclease. Subsequent homology-mediated repair from an episomal donor resulted in XGPRT reconstitution and cellular resistance to a selection antibiotic. Analogously, the repair of chromosomal I-SceI breaks by NHR using another novel reporter plasmid restored XGPRT translation. For p53-null cells, the mean frequency of I-SceI break repair via HR was 5.5 x 10 -4 . The p53-Val135 mutant, which previously has been shown to suppress spontaneous HR by 14-fold employing the same cell system and reporter gene, only caused a 2- to 3-fold suppression of break-induced HR. In contrast, a dramatic effect of p53 on repair via NHR was found. Preliminary sequence analysis indicated that there was at least a 1000-fold reduction of illegitimate repair events resulting in loss of sequence at the break sites. The observed effects were mediated by p53 mutants defective in regulation of the cell-cycle and apoptosis. The main findings were: (1) p53 virtually blocked illegitimate rejoining of chromosomal ends. (2) The suppression of homologous DSB repair was less pronounced than the inhibition of spontaneous HR. We hypothesize that p53 allows to a certain extent error-free homology-dependent repair to proceed, while blocking error-prone NHR. The data support and extent a previous model, in which p53 maintains genomic stability by regulating recombination independently of its transactivation function

  9. Identification and Partial Characterization of Potential FtsL and FtsQ Homologs of Chlamydia

    Directory of Open Access Journals (Sweden)

    Scot P Ouellette

    2015-11-01

    Full Text Available Chlamydia is amongst the rare bacteria that lack the critical cell division protein FtsZ. By annotation, Chlamydia also lacks several other essential cell division proteins including the FtsLBQ complex that links the early (e.g. FtsZ and late (e.g. FtsI/Pbp3 components of the division machinery. Here, we report chlamydial FtsL and FtsQ homologs. Ct271 aligned well with E. coli FtsL and shared sequence homology with it, including a predicted leucine-zipper like motif. Based on in silico modeling, we show that Ct764 has structural homology to FtsQ in spite of little sequence similarity. Importantly, ct271/ftsL and ct764/ftsQ are present within all sequenced chlamydial genomes and are expressed during the replicative phase of the chlamydial developmental cycle, two key characteristics for a chlamydial cell division gene. GFP-Ct764 localized to the division septum of dividing transformed chlamydiae, and, importantly, over-expression inhibited chlamydial development. Using a bacterial two-hybrid approach, we show that Ct764 interacted with other components of the chlamydial division apparatus. However, Ct764 was not capable of complementing an E. coli FtsQ depletion strain in spite of its ability to interact with many of the same division proteins as E. coli FtsQ, suggesting that chlamydial FtsQ may function differently. We previously proposed that Chlamydia uses MreB and other rod-shape determining proteins as an alternative system for organizing the division site and its apparatus. Chlamydial FtsL and FtsQ homologs expand the number of identified chlamydial cell division proteins and suggest that Chlamydia has likely kept the late components of the division machinery while substituting the Mre system for the early components.

  10. A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

    OpenAIRE

    Zhou, Yihua; Xu, Bixiong C.; Maheshwari, Hiralal G.; He, Li; Reed, Michael; Lozykowski, Maria; Okada, Shigeru; Cataldo, Lori; Coschigamo, Karen; Wagner, Thomas E.; Baumann, Gerhard; Kopchick, John J.

    1997-01-01

    Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/...

  11. Compositional Homology and Creative Thinking

    Directory of Open Access Journals (Sweden)

    Salvatore Tedesco

    2015-05-01

    Full Text Available The concept of homology is the most solid theoretical basis elaborated by the morphological thinking during its history. The enucleation of some general criteria for the interpretation of homology is today a fundamental tool for life sciences, and for restoring their own opening to the question of qualitative innovation that arose so powerfully in the original Darwinian project. The aim of this paper is to verify the possible uses of the concept of compositional homology in order to provide of an adequate understanding of the dynamics of creative thinking.

  12. A hidden markov model derived structural alphabet for proteins.

    Science.gov (United States)

    Camproux, A C; Gautier, R; Tufféry, P

    2004-06-04

    Understanding and predicting protein structures depends on the complexity and the accuracy of the models used to represent them. We have set up a hidden Markov model that discretizes protein backbone conformation as series of overlapping fragments (states) of four residues length. This approach learns simultaneously the geometry of the states and their connections. We obtain, using a statistical criterion, an optimal systematic decomposition of the conformational variability of the protein peptidic chain in 27 states with strong connection logic. This result is stable over different protein sets. Our model fits well the previous knowledge related to protein architecture organisation and seems able to grab some subtle details of protein organisation, such as helix sub-level organisation schemes. Taking into account the dependence between the states results in a description of local protein structure of low complexity. On an average, the model makes use of only 8.3 states among 27 to describe each position of a protein structure. Although we use short fragments, the learning process on entire protein conformations captures the logic of the assembly on a larger scale. Using such a model, the structure of proteins can be reconstructed with an average accuracy close to 1.1A root-mean-square deviation and for a complexity of only 3. Finally, we also observe that sequence specificity increases with the number of states of the structural alphabet. Such models can constitute a very relevant approach to the analysis of protein architecture in particular for protein structure prediction.

  13. Model for calculation of electrostatic contribution into protein stability

    Science.gov (United States)

    Kundrotas, Petras; Karshikoff, Andrey

    2003-03-01

    Existing models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this presentation a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi-random distribution, restricted by the protein sequence. The model was tested on two proteins, barnase and N-terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, Δ G( pH), reproduces the experimental data essentially better than the commonly used null approximation (NA). It was demonstrated that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pair-wise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.

  14. Role of phosphatase and tensin homolog deleted on chromosome ten in a rat model of carbon tetrachloride-induced liver fibrosis and the effect of qi-tonifying and blood-activating prescription

    Directory of Open Access Journals (Sweden)

    NIU Xuemin

    2018-01-01

    Full Text Available Objective To investigate the role of phosphatase and tensin homology deleted on chromosome ten (PTEN in a rat model of carbon tetrachloride (CCl4-induced liver fibrosis and the molecular mechanism of action of qi-tonifying and blood-activating prescription in regulating PTEN and inhibiting liver fibrosis. Methods A total of 27 male Wistar rats were randomly divided into three groups, with 9 rats in each group. The rats in liver fibrosis group were treated with CCl4 to establish a model of liver fibrosis, and those in qi-tonifying and blood-activating prescription group were also treated with CCl4 to establish a model and then given a self-made qi-tonifying and blood-activating prescription containing Astragalus membranaceus, Salvia miltiorrhiza, and poria. The rats in the control group were given intraperitoneally injected olive oil. HE staining, Masson staining, and immunohistochemical staining of collagen type I alpha 1 (Col1A1 and collagen type Ⅳ (Col4 were performed to observe the degree of liver fibrosis and collagen deposition; qRT-PCR, immunohistochemistry, and Western blot were used to measure the expression of transforming growth factor-β1 (TGF-β1, PTEN, and downstream genes AKT, mTOR, and p70S6K. A one-way analysis of variance was used for comparison of continuous data between multiple groups and the least significant difference t-test was used for further comparison between any two groups. Results In the liver fibrosis group, liver pathology showed perisinusoidal fibrosis and fibrous tissue proliferation, collagen deposition, and formation of fibrous septum in the portal area; compared with the control group, the liver fibrosis group had significant increases in the mRNA and protein expression of TGF-β1, a significant reduction in the expression of PTEN, and significant increases in the mRNA and phosphorylated protein expression of AKT, mTOR, and p70S6K (all P<0.01. The qi-tonifying and blood-activating prescription group had a

  15. In silico modeling of the yeast protein and protein family interaction network

    Science.gov (United States)

    Goh, K.-I.; Kahng, B.; Kim, D.

    2004-03-01

    Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

  16. Statistical Inference for Porous Materials using Persistent Homology.

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Chul [Univ. of Georgia, Athens, GA (United States); Heath, Jason E. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Mitchell, Scott A. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2017-12-01

    We propose a porous materials analysis pipeline using persistent homology. We rst compute persistent homology of binarized 3D images of sampled material subvolumes. For each image we compute sets of homology intervals, which are represented as summary graphics called persistence diagrams. We convert persistence diagrams into image vectors in order to analyze the similarity of the homology of the material images using the mature tools for image analysis. Each image is treated as a vector and we compute its principal components to extract features. We t a statistical model using the loadings of principal components to estimate material porosity, permeability, anisotropy, and tortuosity. We also propose an adaptive version of the structural similarity index (SSIM), a similarity metric for images, as a measure to determine the statistical representative elementary volumes (sREV) for persistence homology. Thus we provide a capability for making a statistical inference of the uid ow and transport properties of porous materials based on their geometry and connectivity.

  17. Prediction of protein-protein interactions between viruses and human by an SVM model

    Directory of Open Access Journals (Sweden)

    Cui Guangyu

    2012-05-01

    Full Text Available Abstract Background Several computational methods have been developed to predict protein-protein interactions from amino acid sequences, but most of those methods are intended for the interactions within a species rather than for interactions across different species. Methods for predicting interactions between homogeneous proteins are not appropriate for finding those between heterogeneous proteins since they do not distinguish the interactions between proteins of the same species from those of different species. Results We developed a new method for representing a protein sequence of variable length in a frequency vector of fixed length, which encodes the relative frequency of three consecutive amino acids of a sequence. We built a support vector machine (SVM model to predict human proteins that interact with virus proteins. In two types of viruses, human papillomaviruses (HPV and hepatitis C virus (HCV, our SVM model achieved an average accuracy above 80%, which is higher than that of another SVM model with a different representation scheme. Using the SVM model and Gene Ontology (GO annotations of proteins, we predicted new interactions between virus proteins and human proteins. Conclusions Encoding the relative frequency of amino acid triplets of a protein sequence is a simple yet powerful representation method for predicting protein-protein interactions across different species. The representation method has several advantages: (1 it enables a prediction model to achieve a better performance than other representations, (2 it generates feature vectors of fixed length regardless of the sequence length, and (3 the same representation is applicable to different types of proteins.

  18. Polar representation of centrifugal pump homologous curves

    International Nuclear Information System (INIS)

    Veloso, Marcelo Antonio; Mattos, Joao Roberto Loureiro de

    2008-01-01

    Essential for any mathematical model designed to simulate flow transient events caused by pump operations is the pump performance data. The performance of a centrifugal pump is characterized by four basic parameters: the rotational speed, the volumetric flow rate, the dynamic head, and the hydraulic torque. Any one of these quantities can be expressed as a function of any two others. The curves showing the relationships between these four variables are called the pump characteristic curves, also referred to as four-quadrant curves. The characteristic curves are empirically developed by the pump manufacturer and uniquely describe head and torque as functions of volumetric flow rate and rotation speed. Because of comprising a large amount of points, the four-quadrant configuration is not suitable for computational purposes. However, it can be converted to a simpler form by the development of the homologous curves, in which dynamic head and hydraulic torque ratios are expressed as functions of volumetric flow and rotation speed ratios. The numerical use of the complete set of homologous curves requires specification of sixteen partial curves, being eight for the dynamic head and eight for the hydraulic torque. As a consequence, the handling of homologous curves is still somewhat complicated. In solving flow transient problems that require the pump characteristic data for all the operation zones, the polar form appears as the simplest way to represent the homologous curves. In the polar method, the complete characteristics of a pump can be described by only two closed curves, one for the dynamic head and other for the hydraulic torque, both in function of a single angular coordinate defined adequately in terms of the quotient between volumetric flow ratio and rotation speed ratio. The usefulness and advantages of this alternative method are demonstrated through a practical example in which the homologous curves for a pump of the type used in the main coolant loops of a

  19. Parametric representation of centrifugal pump homologous curves

    International Nuclear Information System (INIS)

    Veloso, Marcelo A.; Mattos, Joao R.L. de

    2015-01-01

    Essential for any mathematical model designed to simulate flow transient events caused by pump operations is the pump performance data. The performance of a centrifugal pump is characterized by four basic quantities: the rotational speed, the volumetric flow rate, the dynamic head, and the hydraulic torque. The curves showing the relationships between these four variables are called the pump characteristic curves. The characteristic curves are empirically developed by the pump manufacturer and uniquely describe head and torque as functions of volumetric flow rate and rotation speed. Because of comprising a large amount of points, this configuration is not suitable for computational purposes. However, it can be converted to a simpler form by the development of the homologous curves, in which dynamic head and hydraulic torque ratios are expressed as functions of volumetric flow and rotation speed ratios. The numerical use of the complete set of homologous curves requires specification of sixteen partial curves, being eight for the dynamic head and eight for the hydraulic torque. As a consequence, the handling of homologous curves is still somewhat complicated. In solving flow transient problems that require the pump characteristic data for all the operation zones, the parametric form appears as the simplest way to deal with the homologous curves. In this approach, the complete characteristics of a pump can be described by only two closed curves, one for the dynamic head and other for the hydraulic torque, both in function of a single angular coordinate defined adequately in terms of the quotient between volumetric flow ratio and rotation speed ratio. The usefulness and advantages of this alternative method are demonstrated through a practical example in which the homologous curves for a pump of the type used in the main coolant loops of a pressurized water reactor (PWR) are transformed to the parametric form. (author)

  20. Modeling compositional dynamics based on GC and purine contents of protein-coding sequences

    KAUST Repository

    Zhang, Zhang; Yu, Jun

    2010-01-01

    Background: Understanding the compositional dynamics of genomes and their coding sequences is of great significance in gaining clues into molecular evolution and a large number of publically-available genome sequences have allowed us to quantitatively predict deviations of empirical data from their theoretical counterparts. However, the quantification of theoretical compositional variations for a wide diversity of genomes remains a major challenge.Results: To model the compositional dynamics of protein-coding sequences, we propose two simple models that take into account both mutation and selection effects, which act differently at the three codon positions, and use both GC and purine contents as compositional parameters. The two models concern the theoretical composition of nucleotides, codons, and amino acids, with no prerequisite of homologous sequences or their alignments. We evaluated the two models by quantifying theoretical compositions of a large collection of protein-coding sequences (including 46 of Archaea, 686 of Bacteria, and 826 of Eukarya), yielding consistent theoretical compositions across all the collected sequences.Conclusions: We show that the compositions of nucleotides, codons, and amino acids are largely determined by both GC and purine contents and suggest that deviations of the observed from the expected compositions may reflect compositional signatures that arise from a complex interplay between mutation and selection via DNA replication and repair mechanisms.Reviewers: This article was reviewed by Zhaolei Zhang (nominated by Mark Gerstein), Guruprasad Ananda (nominated by Kateryna Makova), and Daniel Haft. 2010 Zhang and Yu; licensee BioMed Central Ltd.

  1. Modeling compositional dynamics based on GC and purine contents of protein-coding sequences

    KAUST Repository

    Zhang, Zhang

    2010-11-08

    Background: Understanding the compositional dynamics of genomes and their coding sequences is of great significance in gaining clues into molecular evolution and a large number of publically-available genome sequences have allowed us to quantitatively predict deviations of empirical data from their theoretical counterparts. However, the quantification of theoretical compositional variations for a wide diversity of genomes remains a major challenge.Results: To model the compositional dynamics of protein-coding sequences, we propose two simple models that take into account both mutation and selection effects, which act differently at the three codon positions, and use both GC and purine contents as compositional parameters. The two models concern the theoretical composition of nucleotides, codons, and amino acids, with no prerequisite of homologous sequences or their alignments. We evaluated the two models by quantifying theoretical compositions of a large collection of protein-coding sequences (including 46 of Archaea, 686 of Bacteria, and 826 of Eukarya), yielding consistent theoretical compositions across all the collected sequences.Conclusions: We show that the compositions of nucleotides, codons, and amino acids are largely determined by both GC and purine contents and suggest that deviations of the observed from the expected compositions may reflect compositional signatures that arise from a complex interplay between mutation and selection via DNA replication and repair mechanisms.Reviewers: This article was reviewed by Zhaolei Zhang (nominated by Mark Gerstein), Guruprasad Ananda (nominated by Kateryna Makova), and Daniel Haft. 2010 Zhang and Yu; licensee BioMed Central Ltd.

  2. The research methods and model of protein turnover in animal

    International Nuclear Information System (INIS)

    Wu Xilin; Yang Feng

    2002-01-01

    The author discussed the concept and research methods of protein turnover in animal body. The existing problems and the research results of animal protein turnover in recent years were presented. Meanwhile, the measures to improve the models of animal protein turnover were analyzed

  3. Mechanical Modeling and Computer Simulation of Protein Folding

    Science.gov (United States)

    Prigozhin, Maxim B.; Scott, Gregory E.; Denos, Sharlene

    2014-01-01

    In this activity, science education and modern technology are bridged to teach students at the high school and undergraduate levels about protein folding and to strengthen their model building skills. Students are guided from a textbook picture of a protein as a rigid crystal structure to a more realistic view: proteins are highly dynamic…

  4. Completion of autobuilt protein models using a database of protein fragments

    International Nuclear Information System (INIS)

    Cowtan, Kevin

    2012-01-01

    Two developments in the process of automated protein model building in the Buccaneer software are described: the use of a database of protein fragments in improving the model completeness and the assembly of disconnected chain fragments into complete molecules. Two developments in the process of automated protein model building in the Buccaneer software are presented. A general-purpose library for protein fragments of arbitrary size is described, with a highly optimized search method allowing the use of a larger database than in previous work. The problem of assembling an autobuilt model into complete chains is discussed. This involves the assembly of disconnected chain fragments into complete molecules and the use of the database of protein fragments in improving the model completeness. Assembly of fragments into molecules is a standard step in existing model-building software, but the methods have not received detailed discussion in the literature

  5. Modeling and protein engineering studies of active and inactive states of human dopamine D2 receptor (D2R) and investigation of drug/receptor interactions.

    Science.gov (United States)

    Salmas, Ramin Ekhteiari; Yurtsever, Mine; Stein, Matthias; Durdagi, Serdar

    2015-05-01

    Homology model structures of the dopamine D2 receptor (D2R) were generated starting from the active and inactive states of β2-adrenergic crystal structure templates. To the best of our knowledge, the active conformation of D2R was modeled for the first time in this study. The homology models are built and refined using MODELLER and ROSETTA programs. Top-ranked models have been validated with ligand docking simulations and in silico Alanine-scanning mutagenesis studies. The derived extra-cellular loop region of the protein models is directed toward the binding site cavity which is often involved in ligand binding. The binding sites of protein models were refined using induced fit docking to enable the side-chain refinement during ligand docking simulations. The derived models were then tested using molecular modeling techniques on several marketed drugs for schizophrenia. Alanine-scanning mutagenesis and molecular docking studies gave similar results for marketed